Monoclonal antibodies and antisera to intragonadal regulatory protein

ABSTRACT

A protein having a molecular weight of from about 10,000 to 18,000 daltons, isoelectric points of from about pH 4.0 to 6.5 and having the reversible biological effect of inhibiting aromatase activity in a biological system, and antibodies to the protein, modulate follicular development and spermatogenesis and provide for diagnostic tests of gonadal functions.

The United States Government has rights in this invention under ClinicalInvestigator Award HD-00401 and NIH Grant HD-05932.

This application is a continuation-in-part of Ser. No. 06/861,118 filedMay 7, 1986, now abandoned, which in turn is a divisional of Ser. No.06/661,002 filed Oct. 11, 1984, now abandoned, which in turn is acontinuation-in-part of Ser. No. 06/475,416 filed Mar. 15, 1983, nowabandoned; and a continuation-in-part of Ser. No. 06/912,445 filed Sept.25, 1986, now U.S. Pat. No. 4,764,502.

FIELD OF THE INVENTION

This invention relates generally to the field of biochemistry, and moreparticularly to the chemistry and physiology of gonadal function.

BACKGROUND AND SUMMARY OF THE INVENTION

The term gonad refers to the mammalian reproduction system, and includesboth the testis and the ovary. The function of these glands is dependentupon the interrelationship of a number of hormones and proteins, many ofwhich are found, in varying amounts, in both sexes.

The ovary is the essential female reproductive organ in which eggs areproduced. In vertebrates there are commonly two ovaries, suspended fromthe dorsal surface of the broad ligaments, one on each side of theuterus. Adult human ovaries are composed of a fibrous vascular stroma inwhich are imbedded the Graafian follicles, which contain the eggs. Theeggs are discharged by the bursting of the Graffian follicles on thesurface of the ovary and are then immediately received into the mouth ofthe oviduct. Thereafter, the eggs flow through the fallopian tube to theuterus, covered by a mucous membrane known as the endometrium, where thefertilized ovum develops.

The egg cells or ova are periodically matured in the ovaries atintervals of approximately four weeks. At the end of each four weekperiod, one egg reaches maturity and passes into one of the fallopiantubes. The egg descends gradually and remains viable for a short while,and during this time fertilization may take place.

Within the ovary, there is a layer of cells called the germinalepithelium. Here, the potential egg begins its existence and continuesto develop until a primary follicle, i.e., a group of cells isolatedfrom the main layer, is formed around the potential egg. During alifetime, each human ovary forms between 200,000 and 400,000 follicles.Of all these potential eggs only a few develop into mature eggs, most ofthem degenerating. The primary follicle that does not degenerateincreases in size, and the egg cell itself enlarges up to thirty timesits original size.

Other changes occur in the areas adjacent to the follicle. As thefollicle matures, it moves toward the surface of the ovary and when thematuration process is complete, the follicle protrudes from the surface.At this time, ovulation occurs, i.e., the follicle bursts and the egg isexpelled from the surface of the ovary.

The developing follicle produces sex hormones by metabolizingpre-hormones using a series of enzymes: 3β-ol dehydrogenase,17α-hydroxylase, hydroxysteroid dehydrogenase, and aromatase. Aromataseis a central enzyme in the production of sex hormones referred to asestrogens (estradiol, estrone and estriol).

Estrogenic hormones play a particularly important role in both themenstrual cycle and the reproductive cycle. Although 17β-estradiol isthe principal estrogenic hormone, a number of other estrogenicsubstances have been isolated, including estriol and estrone. Thesehormones induce the growth of the vaginal epithilium and secretion ofmucous by the glands of the cervix and initiate the growth of theendometrium.

The corpus luteum, which fills a ruptured Graafian follicle in themammalian ovary, produces at least three hormones, progesterone,17β-estradiol and relaxin. Progesterone acts to complete theproliferation of the endometrium, which was initiated by the estrogenichormones, and to prepare it for the implantation of the ovum.

This reproductive cycle is well regulated as long as the production andsecretion of both the sex hormones from the ovaries and the gonadotropichormones of the pituitary gland are within normal limits. The anteriorlobe of the pituitary, by manufacturing and secreting the gonadotropichormones, controls the production of the sexual hormones in the ovariesand stimulates the development of the reproductive organs and themaintenance of their structure. The ovaries, under control of thegonadotropic hormones, produce the female sexual hormones. In turn, therate of production of gonadotropins by the pituitary is influenced bythe production of sex hormones. The effects are mutual and the twoglands maintain an exact balance in hormone production.

More specifically, the follicle-stimulating hormone (FSH) from thepituitary stimulates the Graafian follicles, which thus produceestrogens. Estrogens not only inhibit FSH production through negativefeedback on the pituitary, but also stimulate the pituitary to increaseits production of luteinizing hormone (LH) through positive feedback.This hormone (LH) in turn brings about ovulation of the Graafianfollicle. After the ova are discharged, the LH stimulates the emptyfollicle, now the corpus luteum, to produce progesterone. This hormonebrings about the changes in the reproductive organs required for thedevelopment of the embryo. The progesterone then partly inhibits thepituitary from producing more LH. Thus, there is no further ovulation.The subsequent fall in progesterone then releases the pituitaryinhibition allowing for the production of FSH to begin the process anew.

When pregnancy occurs, the placenta of the embryo itself produces humanchorionic gonadotropin (hCG), which stimulates the continuing productionof progesterone from the corpus luteum, thus preventing menstruation andstimulating the continuing development of the uterus. This progesteronealso inhibits further ovulation in spite of the presence of the hCG fromthe placenta.

The gonadotropic hormones have been determined to be proteins, withvariable amounts of carbohydrates, and their structures are known. Themolecular weight of human LH is about 26,000, and that of human FSH isabout 30,000. The cellular response to gonadotropic hormones istranslated through cellular receptors. These receptors are locatedwithin the cell membrane and are specific for each gonadotropin, thus,LH only activates LH receptors and FSH only activates FSH receptors.

Non-abortifacient means for the avoidance of pregnancy include oralcontraceptive medications which contain estrogen and/orprogesterone-like steroidal sex hormones. These medications, by raisingthe level of sex hormones in the blood stream, generate a cervicalmucous which is hostile to spermatazoa. With increased levels of suchhormones the endometrium tends to resist implantation of the fertilizedova. Further, the excess hormones provided by oral contraceptivesdirectly inhibit the release of LH and FSH by the pituitary. As such,the ovarian cycle is disrupted and ovulation does not occur. Thecomplications of such steroidal contraceptive medications, e.g., nausea,vomiting, weight gain, hypertension and tumor stimulation, adverseeffects on calcium and phosphate metabolism, are well known and need notbe discussed at length. However, these side effects result not only fromthe abnormally high levels of steroidal hormones in the blood stream,but from disruption of the hormonic homeostatis of the organism, i.e.,the cycle of hormone adjustment between the ovary and the pituitarygland.

Accordingly, it has been a desideratum to provide a contraceptivemedication which permits the regulation of the ovarian process withoutan accompanying disruption of the hormonic homeostatis of the organism.

While sex hormones are commonly referred to as male (androgenic) andfemale (estrogenic) hormones, these substances, including theproteinaceous substances associated therewith, are each important in theregulation of both the male and the female reproductive systems. Forexample, the luteinizing hormone (LH) and follicle stimulating hormone(FSH) play important roles in the regulation of the testis. Thesegonadotropins are synthesized and released in the male pituitary underthe regulation of a hypothalamic peptide (luteinizing hormone releasinghormone) which is synthesized in the hypothalamus. LH binds to thesurface receptors on the Leydig cells and promotes increasedtestosterone synthesis. It should be noted that testosterone synthesisis also required by the prevailing estradiol concentration, with highestradiol levels decreasing testosterone synthesis.

Spermatogenesis is a complex event where primitive germ cells (thespermatogonia) proceed through multiple cell divisions, in an orderlyprogression from spermatogonia to spermatocyte to spermatid, in order toform mature spermatozoa. In this progression, the intergenderalcommunality of the regulating hormones and proteins in the gonads isdemonstrated by the fact that estrogen is a necessary component in theconversion of spermatogonia to spermatocytes. Further, the enzymearomatase is a central enzyme in the conversion of androgens toestrogens. It should be noted that aromatase is a paracrine(intragonadal) enzyme that has been shown to be critical to theregulation of fertility in both the male and female gender of mammalianspecies.

It has also been a desideratum to provide a facile method for theregulation of fertility in both the male and female gender of mammalianspecies. It should be understood that the regulation of fertility, asemployed herein, refers to a method and/or a composition of matterassociated therewith, which enables either an increase or a decrease inmammalian fertility, that is, fertility control as opposed to merecontraception.

The present invention accomplishes the foregoing objectives by providinga protein moiety which enables the intragonadal modulation of the levelof aromatase in a mammalian host and thus can regulate the maturation ofovarian follicles and the production of viable ova and regulatespermatogenesis and the production of mature spermatozoa withoutdisturbing the normal level of sex hormones in the body, and permit theevaluation and diagnosis of gonadal function and dysfunction.

According to the invention a purified, non-steroidal proteinaceousmaterial or moiety having a measurable molecular weight of up to about20,000, an isoelectric point of from about pH 3.5 to about pH 7.0 isprovided, and is characterized by having the biological effect ofinhibiting aromatase activity as defined by the extent of the conversionof androgens to estrogens. The protein inhibits intragonadal aromataseactivity, modulates the intragonadal activity of 3β-ol dehydrogenase,inhibits the development of granulosa cell LH receptors and prevents thematuration of mature ova and the production of mature spermatozoa. Theterm protein moiety, as used herein, refers to a protein, proteins orfunctional operative groups thereon which produce the described results.The production and activity of the protein moiety is interspecies, andis effective in both monotocous and polytocous mammals. Further, methodsare provided for the isolation, purification and production of theprotein, and for the use thereof in the regulation of fertility control.

In another aspect of the invention, antibodies are provided whichinhibit the natural production of the protein with a correspondingincrease in aromatase activity resulting in the promotion of folliculardevelopment, ovum maturation and spermatogenesis. Thus, mammalianfertility may be controlled substantially independently of exogenous sexhormones and without modulating extragonadal hormone levels. In yetanother aspect of the invention, methods are provided for the use ofantibodies to the protein for the quantification of the level of theprotein in body fluids, thus permitting the evaluation and diagnosis ofgonadal function and dysfunction.

For convenience, the intragonadal and follicular regulating protein isreferred to herein as FRP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the elution profile of a preovulatory patient's ovarianvenous effluent after partial purification using Sephadex G-50 superfinechromatography with absorbance measured at 254 nm. The responses, bybioassay of hypophysectomized, twenty-three day old, DES-treated ratovaries receiving hMG therapy to aliquots of the Sephadex G-50 fractionsin terms of ovarian weight and serum 17β-estradiol concentrations areoverlayed. Fractions eluting with a V_(e) /V_(O) of 1.42-1.55 activelyinhibited both parameters.

FIG. 2 shows the dose-response of active Sephadex G-50 fractions (V_(e)/V_(O) =1.42-1.55) in bioassay of hypophysectomized, twenty-threeday-old, DES-treated rat ovaries receiving hMG treatment. Insert, Rankitanalysis to determine the central tendency for ovarian weight inhibitionat each dose tested.

FIG. 3 shows a compilation of the active Sephadex G-25 fractions (V_(e)/V_(O) =1.20-1.25) pooled and developed by ampholyte displacementchromatography. Fractions corresponding to a pH of 6.1-6.5 manifestinhibitory activity in excess of 50% tested in the bioassay withhypophysectomized, twenty-three-day-old, DES-treated rat ovariesreceiving hMG therapy (ovarian weight). Ampholyte displacementchromatography of the fractions with follicle-inhibiting activityobtained on Sephadex G-25 gel filtration (V_(e) /V_(o) =1.20-1.25).

FIG. 4: K_(av) of the active portion of preovulatory ovarian venouseffluents from patients 1-3 eluted through a Sephadex G-50 superfinecolumn calibrated with molecular weight standards. The molecular weightsestimated in this manner were 14,000-16,500 for patient 1, 14,000-17,000for 2 and 16,000-18,000 for patient 3 .

FIG. 5: shows an elution profile of the 10-55% SAS cut of humanfollicular fluid, dialyzed and developed through Orange A Dye-Matrix(1.5M KCl), passed through Sephadex G-50. Fractions (2 ml) were testedin the LH-FSH-stimulated hypophysectomized, immature, DES-treated ratfor inhibition of ovarian weight, and serum estradiol concentration(mean±SEM). Fractions with molecular weight corresponding to12,000-18,000 contained inhibitory activity.

FIG. 6. Isoelectric focusing chromatogram of pooled human follicularfluid after SAS precipitation (10-55%), dialysis and Orange A Dye-Matrixchromatography (0.5M KCl eluent). Fractions corresponding to anisoelectric point of pH 3.5-4.5 contained inhibitory activity of ratovarian weight and trunk estradiol (not shown) in the bioassay aftergonadotropin challenge.

FIG. 7: Ampholyte displacement chromatogram of pooled human follicularfluid (hFF) after SAS precipitation (10-55%), dialysis (10,000 molecularweight) and Orange A Dye-Matrix chromatography (1.5M KCl eluent).Fractions corresponding to an isoelectric point of pH 3.5-4.5 and pH6.5-7.0 contained inhibition of rat ovarian weight (mean±SEM) in thebioassay after gonadotropin challenge.

FIG. 8: Representative high performance size exclusion liquidchromatogram (TSK 3000 analytical column) of the Orange A-bound fractionof the 10-55% SAS hFF. Fractional retention times correspond to thefollowing approximate molecular weight ranges: 21.0-23.0minute, >100,000; 23.0-25.0 minutes, 100,000; 25.0-28.5 minutes,70,000-100,000; 28.5-32.2 minutes, 40,000-70,000; 32.2-34.0 minutes,30,000-40,000; 34.0-40.0 minutes, 18,000-30,000; 40.0-45.0 minutes,5,500-18,000; 45.0-49.0 minutes, 2,500-5,500; 49.0-53.0 minutes, <2,500.

FIG. 9: Representative high performance liquid chromatogram (TSK 3000preparative molecular weight exclusion column) of the 10-55% SASfraction of human follicular fluid (hFF).

FIG. 10: Bioassay results of HPLC fraction of Orange A bound hFF.Inhibition of LH-FSH-induced ovarian weight augmentation in immature,hypophysectomized, DES-treated rats (mean±SEM) was evident in thosetreated with the 5,500-18,000 molecular weight fractions (no. 7, n=3rats at each level).

FIG. 11: A polyacrylamide gel, with sodium dodecyl sulfate, of thefractions shown in FIG. 8 after HPLC.

FIG. 12: Isoelectric focusing chromatogram of dialyzed (10,000 mw),10-55% saturated ammonium sulfate fraction of pooled porcine follicularfluid (5 ml) after elution (0.5M KCl) from an Orange A dye matrixcolumn. Maximum ovarian weight inhibition (hatched bars) in immature,hypophysectomized, DES-treated rats by eluent fractions were found inthe 3.7-4.5 pH range (X±SEM of rats/fraction).

FIG. 13: Dose response relationship: porcine follicular fluid fractionswhich contained inhibitory activity in the rat ovarian weightaugmentation bioassay recovered from isoelectric focusing (ph 4.0-4.5)after Orange A Dye Matrix elution (0.5M KCl) and dialysis (10,000 mw) of10-55% saturated ammonium sulfate. Insert is Rankit analysis of ovarianweights from dilutions tested, the O intercept of which was plotted onthe dose reponse curve.

FIG. 14: High performance liquid chromatogram (gel permeation) of theOrange A-bound fraction of 10-55% saturated ammonium sulfate of porcinefollicular fluid recovered from Sephadex G100 elution (Ve/Vo 1.3-1.7)Retention times correspond to the following approximate molecular weightranges: 8 min: 100,000-74,000; 8-31 min: 74,000-36,000; 31-36 min:36,000-18,000; 36-39 min: 18,000-12,000; 39-43 min: 12,000-5,800.

FIG. 15: Chromatofocusing by hydrogen ion exchange chromatography of theOrange A-bound fraction of the 10-55% saturated ammonium sulfatefraction of porcine follicular fluid.

FIG. 16: Bioassay results of high performance liquid chromatographyfraction of Orange A-bound porcine follicular fluid after separation bySephadex G-100 chromatography (Ve/Vo 1.3-1.7). Inhibition of LH/FSHinduced ovarian weight augmentation (X±SEM) in immature,hypophysectomized, DES-trated rats (n=6/fraction tested) was measuredafter treatment with 2 ml of HPLC eluents which correspond to thefollowing approximate molecular weights: 1: 100,000-74,000; 2:74,000-36,000; 3: 36,000-18,000; 4: 18,000-12,000; 5: 12,000-5,800.

FIG. 17: Aromatase activity of rat granulosa cells derived fromimmature, hypophysectomized, DES-treated rats (n=6/fraction tested)which received LH/FSH stimulation and injection of high performanceliquid chromatography fractions (2 ml) of extracted porcine follicularfluid (X±SEM). HPLC fractions corresponded to the following approximatemolecular weights: 1: 100,000-74,000; 2: 74,000-36,000; 3:36,000-18,000; 4: 18,000-12,000; 5: 12,000-5,800.

FIG. 18: Binding of FSH to granulosa cells collected from the immature,hypophysectomized, DES-treated rats (n=6) which received LH/FSH (2 IU)and extracted porcine follicular fluid after Sephadex G-100 separation(Ve/Vo 1.3-1.7) (2 ml). Specific binding of rFSH to granulosa cells wasdetermined by incubating three concentrations of labeled rFSH in thepresence and absence of excess unlabeled rFSH.

FIG. 19: Effect of extracted hFF and respective granulosa cell culturemedia (twenty-four, forty-eight and seventy-two hours) on the inhibitionof LH/FSH (2 IU)-stimulated immature, hypophysectomized, DES-treated ratovarian weight augmentation and serum estradiol secretion (2 ml/rat).Each value represents the mean±SE of three rats. These data indicatethat GRP is secreted by the granulosa cells.

FIG. 20: Effect of human granulosa cell culture media (2 ml/rat) on theinhibition of ovarian weight augmentation and serum estradiol responsesto LH/FSH stimulation in the immature, hypophysectomized, DES-treatedrat. Each value represents the mean±SE of nine rats. Granulosa cellswere aspirated from three patients (no. 5-7) who underwent clomiphene(150 mg/day, menstrual cycle days 3-7) and hCG (4000 IU, 36 hours beforeaspiration) treatment.

FIG. 21: Binding of FSH to rat granulosa cells collected from theHIFR-hMG bioassay of non-LH/FSH-stimulated human granulosa cell culturemedium (see FIG. 20). Specific binding of rat FSH (rFSH) to granulosacells was determined by incubating three concentrations of labeled rFSHin the presence and absence of excess unlabeled rFSH.

FIG. 22: Aromatase activity of rat granulosa cells derived from theHIFR-hMG bioassay of human granulosa cell culture medium (see FIG. 20,non-LH/FSH-stimulated cultures). Control determinations were performedon rat granulosa cells collected from HIFR-hMG-treated rats which didnot receive culture medium injections. Inhibition of relative estrogenproduction by rat granulosa cells in the presence of 10⁻⁷ Mandrostenedione was seen throughout all four days of human granulosacell culture medium treatment.

FIG. 23: Inhibition of serum estradiol levels in the peripheral trunkblood of immature, hypophysectomized, diethyl stilbesterol and FSHtreated rats (FSH control) by: ovarian vein serum from FSH treatedbaboons before (pre clomid+FSH) and after (clomid+FSH) clomiphenecitrate therapy; bovine testis extract, (BTE+FSH); and bovine testisextract which binds to matrix Gel Orange A and which elutes with 0.5MKCl+TRIS (BTE-OAB+FSH).

FIG. 24: Inhibition of FSH stimulated (FSH control) porcine granulosacell aromatase activity measured by determination of estradiolconcentrations in spent culture media after incubating the cells withthe following reagents for 24 hours followed by androstenedione (10⁻⁶ M)for three hours: ovarian vein serum from clomiphene citrate treatedbaboons (+clomid); bovine testis extract (+bTEC); bovine testis extractwhich binds to dyematrix Orange A and elutes with 0.5M KCl+TRIS(+bTE-OAB).

FIG. 25: Dose-response inhibition of porcine granulosa cell aromataseactivity by FRP isolated from human follicular fluid. Aromatase activitywas determined in triplicate determinations with granulosa cells (10⁶/culture), androstenedione (10⁻⁶ M) and pFSH (2ng) co-cultured for threehours.

FIG. 26: Correlation of follicular fluid inhibin activity (inhibition ofspontaneous rFSH release by pituicytes) and gonadal regulatory proteinactivity (% aromatase inhibition) in regularly menstruating women eitheruntreated or after receiving HMG or clomiphene therapy. A statisticallysignificant correlation (r=0.650, p<.05) was apparent between inhibinand follicular protein activities in follicular fluid from untreatedpatients only, indicating that inhibin and FRP are not the same protein.

FIG. 27: Lineweaver-Burke plot of FRP (250,500, 1000 ug/ml) inhibitionof human placental microsomal aromatase activity in vitro.

FIG. 28: Dixon plot of FRP inhibition of placental microsomal aromataseactivity in vitro.

FIG. 29: Levels of progesterone (X⁻ ±SEM) produced by cell-freemicrosome enriched preparations from human granulosa cells obtainedduring in vivo hyperstimulation (clomiphene citrate +hMG+hCG) from womenparticipating in an in vitro fertilization protocol. Cells were treatedin vitro with or without hMG and a preparation of FRP termed follicleregulatory protein (N=5 cycles, 3-7 follicles per cycle).

FIG. 30: The effects of FRP and pFSH on 3-beta-ol-dehydrogenaseactivity, after 48 hours of pre-incubation, expressed as ng ofprogesterone (±SEM) produced per 100,000 cells per 24 hours. Total cellculture time equals 72 hours.

FIG. 31: Time course of FRP inhibition of Gpp (NH)p (10 uM)activated-adenylate cyclase. Adenylate cyclase activity was determinedin porcine granulosa cell membranes after incubation of the cells withFSH (3.3 uM), FRP designated follicle regulatory protein (500 μg, o) orFSH (3.3 uM) and FRP (500 μg, 00) for the indicated time. Pointsindicate the mean of three values and the bars indicate the standarderror.

FIG. 32: Effects of FRP on Gpp(NH)p activated adenylate cyclase. CyclicAMP formation by porcine granulosa cell membranes was measured after athirty minute incubation with (Δ, 3.3 um) or without (+) FSH in thepresence of varying concentrations of FRP. Each point represents themean of three determinations and the bars indicate the standarddeviation.

FIG. 33: Composite (X±SEM) of serum LH, FSH, estradiol, and progesteronelevels from 2 rhesus monkeys which received twice daily injections (3mg, intramuscularly) (cycle days 1-4) of FRP purified from porcinefollicular fluid which does not contain inhibin F activity. Barindicates menses. Shaded area in each panel is the 95% confidenceintervals for the respective hormone levels determined for untreated,normally cycling monkey. Data have been synchronized to the LH surge(day 0).

FIG. 34: Composite (X±SEM) of serum LH, FSH, estradiol, and progesteronelevels from 2 rhesus monkeys which received twice daily injections (3mg, intramuscularly) of FRP partially purified from porcine follicularfluid which does not contain inhibin F activity. Bars indicate menses.Shaded area in each panel represents the 95% confidence intervals forthe respective hormone levels determined for untreated normally cyclingmonkeys.

FIG. 35: levels of FRP in the urine of twelve regularly menstruatingwomen over time, the simultaneously-obtained serum levels of LH, FSH,estradiol and progesterone, and the ultrasonongraphic determination ofthe largest follicle diameter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The embodiment hereinafter set forth relates to the isolation andpurification of the intragonadal protein moiety and its use in theinhibition of the activity of the aromatase enzyme, modulation of 3β-oldehyrogenase activity, regulation of mature ova and sperm formation andthe production of the protein moiety by cell cultures.

The protein moiety is isolated from the ovary or the testis by methodssuch as salt fractionation and dialysis, and chromatography. Granulosaor Sertoli cells may be cultured to produce substantial amounts of theprotein moiety, which is then similarly purified.

Generally, gonadal fluid may be obtained by compression of appropriatemammalian gonads, and purified by chromatographic separation to yieldFRP, although initial salt fractionation and subsequent electrophoreticseparation vastly improve the separation procedure.

More specifically, FRP may be extracted from biological fluids includingovaries, testis, rete testis fluid, lymph, blood, and ovarian follicularfluid from mammalian species. To illustrate, a preferred process for theseparation and purification of FRP as set forth in the Examples issummarized as follows:

1. Biological fluid is mixed with saturated ammonium sulfate to achievea 30-55% concentration.

2. The precipitate from step 1 is resuspended in TRIS buffered saline(20 mM, pH 7.5) and dialyzed against 3 changes of distilled water (36hours, 4° C.) to remove the remaining ammonium sulfate.

3. The retentate from step 2 is passed through a hydroxylapatite columnwhich is subsequently washed with 2 column volumes of TRIS bufferedsaline (20 mM, pH 7.5). The column is then eluted in a stepwise fashionwith 0.1mM phosphate buffer followed by 0.5M phosphate buffer.

4. The eluent from the 0.5M phosphate buffered saline is passed througha Sephacryl G-200 column and eluted with TRIS buffer (20 mM, pH 7.5).

5. The eluent from step 4 which elutes in the <40,000 molecular weightrange is charged onto a Matrix gel Orange A column which was previouslyequilibrated with TRIS buffer (20 mM, pH 7.5). The Matrix gel Orange Acolumn is washed with 3 column volumes of TRIS buffer and then elutedwith TRIS buffer (20 mM, pH 7.5) to which has been added 0.5M KCl.

6. The TRIS+0.5M KC1 eluant from step 5 after dialysis is charged onto ahydrogen ion exchange column previously equilibrated with imidazolebuffer (0.25 M, pH 7.4). The column is eluted with buffer over the pHrange 7.4 to 3.6.

7. The eluent fractions corresponding to about pH 7.4 to pH 3.6 fromstep 6 are eluted through a TSK 3000 column with TRIS buffer (pH 7.5, 20mM) by high performance liquid chromatography. The fractioncorresponding to 35 to 37 minutes in FIG. 8 (analytical column) and 77.8to 78.0 minutes in FIG. 9 (preparative column) contain FRP.

8. The TRIS+0.5M KCl eluent from step 5 is layered on an agarose gel orSephadex G-75 column which have been previously charged with ampholines(pH 3-8). A current is passed through the support matrix of sufficientlength and wattage to allow equilibration of proteins at theirisoelectric points. The fractions recovered from these electrophoreticseparations corresponding to isoelectric points of from pH 3.5 to pH 7.0contain FRP.

9. The TRIS+0.5M KCl eluent from step 5 can be charged onto ahydrophobic reverse phase column. The column can be eluted withincreasing concentrations of organic reagents (acetonitrile,trifluroacetic acid). FRP can be separated from the other components ofstep 5 eluent in this fashion using differential solubility to organicsolvents.

The biological effect of the moiety was assessed in laboratory animalsby determining variations in ovarian weight and analyzing body fluidsfor steroid hormones and enzyme activities. Aromatase inhibition wasdetermined by incubating granulosa cells in the presence of a substrateand FRP and measuring the amount of estrogen formed. The 3β-oldehydrogenase was found to be modulated by incubating granulosa cells inthe presence of a substrate and GRP and measuring the amount ofprogesterone formed. The addition of labelled LH and FSH to in vitrogranulosa cell cultures showed a decrease in LH receptors in the cells,and no change in FSH receptors grown in the presence of FRP.

Regularly ovulatory primates were exposed to FRP and in all instancesthe ovarian cycle was modified, along with follicular growth, asevidenced by estrogen and progesterone levels, without the concomitantalteration of LH and FSH levels. In subsequent cycles, the primates hadtimely menstrual onset and showed no toxic treatment effects. Maleanimals were injected with FRP and exhibited reduction ofspermatogenesis.

Pharmaceutically effective amounts may be administered to individualsorally, by injection, membrane absorption or other means which will beapparent to those skilled in the art. A particularly advantageous methodof administration comprises the introduction of an effective amount ofthe moiety to the absorbent mucous membranes.

Since the FRP inhibits aromatase and normal follicle maturation, itplays a central role in the ovulatory process. Accordingly blockage ofthe GRP activity allows for the maturation of multiple follicles toovulatory status. The protein may be coincubated with hybrid cells invitro to produce antibodies to proteins from different species. Theantibody produced by the hybrid cultures and collected by chromatographymay be injected into females thus blocking the action of the follicularprotein and allowing for development and eventual ovulation of one ormore follicles.

Agricultural uses of a similarly prepared antibody allow for thematuration of multiple follicles in livestock which could be collected,fertilized in vitro and reinserted into a properly prepared surrogate.FRP antibodies may also be employed to quantify the level of existingFRP in body fluids for a wide variety of diagnoses.

EXAMPLES

The following examples will describe in detail the identification,separation, activity and effects of the purified intragonadal regulatoryprotein, according to the following outline:

I. Identification of the Sources and Physical Characteristics of FRP.

A. Ovarian Venous Blood (Example One).

B. Human Follicular Fluid (Example Two).

C. Porcine and Bovine Follicular Fluid (Example Three).

D. Granulosa Cells and Cell Culture (Example Four).

E. Testis Fluid (Example Five).

F. Sertoli Cells and Cell Culture (Example Six).

II. Biological Activity

A. Aromatase Inhibition (Example Seven).

1. Non-Competitive Inhibition (Example Eight).

B. Modulation of 3β-ol-dehydrogenase Activity (Example Nine).

C. Inhibition of LH-hCG Receptor Formation (Example Ten).

D. Inhibition of FSH Augmented Adenylate Cyclase Activity (ExampleEleven).

E. Reduction of Follicular Atresia (Example Twelve).

III. Whole Animal Studies

A. Inhibition of Primate Ovarian Cycles (Example Thirteen).

B. Inhibition of Spermatogenesis (Example Fourteen).

IV. Preparation of FRP Antibodies (Example Fifteen) and use indiagnostic procedures (Example Sixteen).

I. Identification of the Sources and Physical Characteristics of FRPEXAMPLE ONE Identification of Follicular Inhibitory Protein(s) inOvarian Venous Blood

Ovarian venous blood (5 ml) was collected from six women (aged 26, 28,31, 36, 37 and 41 years) undergoing laparotomy for indications notrelated to ovarian dysfunction on days 12-14 after the onset of the lastmenstrual period. Patients 1-3 maintained regular menstrual cycles,while patients 4-6 were anovulatory, as evidenced by oligomenorrhea anda lack of large antral follicles in the ovarian cortex. A 25-gaugeneedle was inserted into the venous drainage within theinfundibulo-pelvic ligament, and free-flowing blood was aspirated. Inaddition, the locus of the preovulatory follicle was determined bydirect visual inspection. Peripheral serum (10 ml) was collectedconcurrently from an antecubital vein. Serum was separated bycentrifugation (800×g for fifteen minutes) of the clotted specimen andstored frozen (-35° C.) until fractionation. Concentrations of17β-estradiol in ovarian (1340, 886, and 470 pg/ml) and peripheral (248,261, and 201 pg/ml) venous samples collected from patients 1, 2 and 3,respectively, were consistent with the preovulatory 17β-estradiol levelsreported for normal women. In addition, comparable samples fromanovulatory patients 4-6 contained low levels of 17β-estradiol in bothovarian (200 pg/ml) and peripheral (50 pg/ml) sera. Slowly thawed serumwas fractionated by the dropwise addition of an equal volume ofsaturated ammonium sulfate under persistent agitation at 4° C. Aftertwelve hours, the precipitate was resuspended (2:1, vol/vol) with 10%ammonium sulfate. After twelve hours of additional agitation andcentriguation (3000×g for 30 minutes), the supernatant was dialyzed with10,000 molecular weight exclusion membranes against Dulbecco'sphosphate-buffered saline (PBS; 0.025M; pH 6.8) for 16 hours. Theretentate was passed through a Concanavalin A-linked Sepharose 4B (ConA) column (5 ml; Pharmacia, Piscataway, New Jersey) which was washedwith 5 vol 0.5M NaCl, pH 7.4, then further eluted with 0.5Mα-methyl-D-mannoside in PBS at a flow rate of 20 ml/h at 4° C.

Additional fractionations were performed where indicated on a SephadexG-25 (superfine) column (1.6×50 cm; Vo=60 ml; 5 ml/h; 4° C.) with PBS.All Sephadex molecular weight sieving was performed using a reverse flowtechnique. Both Sephadex G-50 and G-25 were prepared according to theinstructions of the manufacturer and equilibrated in the elution buffer.To increase resolution, the smallest of the Sephadex beads were removedby direct pipetting of the surface before degassing and column packing(10 mm H₂ O). Elution profiles were determined using an ISCO absorbancemeter at 254 nm.

The activity was assessed in twenty-three-day-old Sprague-Dawley rats(45-55 g) 2 days after hypophysectomy which were kept at 25° C. withintervals of fourteen hours of light and ten hours of darkness. Animalswere caged in groups of three and given rat chow and water ad libitum.Silastic implants containing DES (diethylstilbestrol) were prepared asfollows. Ten grams of Silastic (TM) polymer were mixed with 3 g DES forthirty minutes at 16° C.; thereafter, four drops of stannous octoatecatalyst were added, with an additional ten minutes of mixing. Thematerial was passed through a Luer-lock syringe (id, 1 mm) into asteaming (95° C.) 0.9% NaCl water bath and annealed for two hours.DES-containing Silastic implants (1×5 mm) were inserted sc in thehypophysectomy incision forty-eight hours before assay. The assay designconsisted of three rats at each dose of reference preparation andunknown. Forty-eight hours after hypophysectomy, animals were giveneither varying concentrations of gonadotropin (LH:FSH, 1:1) dissolved in0.15M NaCl with 1% bovine serum albumin and/or equal volumes of testfractions in two divided daily doses. Twenty-four hours after theinitial injection, animals were sacrificed by decapitation, and ovarieswere removed, trimmed and weighed on a Roller-Smith balance. Rat trunkserum 17β-estradiol determinations were performed by methods describedin Goeblesmann et al., in Leprow et al (Eds.) Vasectomy: Immunologicaland Pathologic Effects in Animals and Man, Academic Press, New York, p.165. Control determination of chromatography fractions containinginhibitory activity was performed by heating (56° C.; for thirtyminutes) or trypsin digestion (20 mg/100 ml) of representative samplesfor 4 hours.

FIG. 1 compares absorbency, at 254 nm, of the Sephadex G-50 fractionsfrom a preovulatory ovarian venous sample of patient 1 to the bioassayresults of the sample, as determined by rat ovarian weight and serum17β-estradiol concentrations. An initial peak rose to 28 absorbencyunits, followed by a gradual downward slope, with the emergence of asmaller second peak (V_(e) /V_(o) =1.42-1.55). When these eluents weretested in the bioassays, the combined rat ovarian weights ranged from57-100 mg, and rat serum 17β-estradiol levels ranged from 70-230 pg/mlthroughout the initial fractions. Thereafter, fractions with a Ve/Vo of1.42-1.55 corresponded to an inhibition of hMG-induced ovarianstimulation in the bioassay, as evidenced by a decrease in ovarianweight (59±0.5 mg) and a significant (P<0.01, by paired t test) decreasein serum 17β-estradiol to levels less than 25 pg/ml. As a consequence,these fractions were pooled and processed for dose response activity.Peripheral and ovarian venous blood collected from the ovarycontralateral to the site of ovulation in patient 1 demonstrated similarG-50 elution profiles (data not shown). However, when representativefractions were tested by bioassay, no reduction in ovarian weight orserum 17β-estradiol was found. Further, ovarian venous bloodpreparations from the anovulatory patients also failed to suppress theresponse of the ovaries to hMG stimulation. However, ovarian venous serafrom the ovulatory ovary of patients 2 and 3 had a similar Sephadex G-50elution profile. Fractions with a V_(e) /V_(o) of 1.48-1.60 suppressedthe response of rat ovarian weight (57.4±2.1 vs. 81.2±4.5 mg; P<0.05)and serum 17β-estradiol concentrations (25 vs. 68-120 pg/ml; P< 0.01) tohMG stimulation. When active fractions from the G-50 eluents of patients1-3 were treated or trypsin digested, they lost their ability tosuppress ovarian weight or 17β-estradiol secretion in response to hMGstimulation.

FIG. 2 depicts the dose-response curve of ovarian weight suppression inthe DES-treated rat ovaries by active Sephadex G-50 fractions (V_(e)/V_(o) =1.42-1.55) derived from patient 1. Analysis of the rat ovarianweight and serum 17β-estradiol concentrations revealed (insert: centraltendency ovarian weights), a linear dose-response pattern. When thesesame fractions were treated with heat or trypsin, no suppression ofovarian weight was present.

The isoelectric point of active fractions eluted from the Sephadex G-50column was estimated by ampholyte displacement chromatography. Pooledaliquots (1 ml) of active fractions were layered on a PolybufferExchanger 94 (25 ml; equilibrated to pH 7.4 with 0.025M Imidazole HCl)column (0.6×30 cm). Fractions were eluted with Pharmalyte.sup.(TM)Polybuffer.sup.(TM) 74-HCl adjusted to pH 4.0 with HCl 1N at 10 ml/h at4° C. Fractions that eluted at a pH greater than 7.4 were collected andrechromatographed in the same Pharmalyte column reequilibrated to pH 9.4with ethanolamine 0.025M and eluted with Polybuffer 96 adjusted to pH6.0 with 1N KOH.

FIG. 3 compiles the active Sephadex G-25 fractions (V_(e) /V_(o)=1.20-1.25) pooled and developed by ampholyte displacement chromatogramsfrom patient 1 for elution ranges pH 9-4. The bioassay results fromovarian weight and serum 17β-estradiol concentrations fromrepresentative fractions suggested that the isoelectric point of theactive Sephadex G-50 eluents from patient 1 were between pH 6.2-6.5.

Fractions corresponding to a pH of 6.1-6.5 manifest inhibitory activityin excess of 50% tested in the bioassay with hypophysectomized,twenty-three-day-old, DES-treated rat ovaries receiving hMG therapy(ovarian weight). Ampholyte displacement chromatography of the fractionswith follicle-inhibiting activity was obtained on Sephadex G-25 gelfiltration fractions with V/V_(o) =1.20-1.25.

FIG. 4 depicts the K_(av) values for the molecular weight standards andactive Sephadex G-50 fractions from patients 1-3. Estimations ofmolecular weight ranged from 14,000-16,500 for patient 1, from14,000-17,000 for patient 2, and from 16,000-18,000 for patient 3.

Thus, it is seen that at least one protein which suppresses the follicleresponse to gonadotropins is secreted by the preovulatory ovary.Specifically, a heat- and trypsin-labile substance is secreted directlyinto the venous drainage from the ovary containing the dominant folliclewhich suppresses the follicular response to gonadotropins. That thisprotein is not secreted in large amounts by anovulatory ovaries wasevidenced by the failure of the bioassay to detect inhibitory activityin the venous drainage of the contralateral ovary of patients 1-3 aswell as the ovarian venous effluents from three anovulatory women. Thispotential intra- and/or interovarian regulator of folliculogenesismediates dominance of the preovulatory follicle by an active process,such that after the selection of the dominant follicle, the gonadotropinresponsitivity of other follicles on the same and contralateral ovariesis suppressed.

EXAMPLE TWO Identification of Follicular Regulatory Protein(s) in PooledHuman Follicular Fluid

To evaluate the role of nonsteroidal, follicular fluid proteins infolliculogenesis, the 10-55% saturated ammonium sulfate fraction ofpooled human follicular fluid was dialyzed against 0.025M Tris/HCl (pH7.5) using 10,000 molecular weight exclusion membranes, then passedthrough agarose immobilized textile dye. Activity was determined by testfraction inhibition of human menopausal gonadotropin (2 U human LH/FSHday) induced ovarian weight gain, and serum estradiol increases inhypophysectomized, diethylstilbesterol-treated, twenty-five-day-oldfemale rats.

Specifically, human follicular fluid was aspirated from regularlymenstruating women (aged twenty-five to thirty-five years) undergoingovarian hyperstimulation during participation in an in vitrofertilization protocol by treatment with clomiphene citrate (150 mg/dayfor five days, beginning three-eight days after onset of spontaneouslyoccurring menses) and hCG (5000 IU forty-eight hours before aspiration).Sera were collected daily and estrogen concentrations were determined.When serum estrogen concentrations exceeded 800 pg/ml, patientsunderwent laparoscopy for aspiration of follicles in excess of 20 mm indiameter. The follicular aspirate was immediately centrifuged (800×g),the granulosa cells were removed and the aspirate was then frozen (-57°C.). Follicular aspirates from twenty such patients were pooled andprovided the hFF used throughout this study.

Pooled hFF was slowly thawed and fractionated by dropwise addition of anequal volume of saturated ammonium sulfate (SAS) during persistentagitation at 4° C. After a twelve hour incubation at 4° C., theprecipitate was pelleted, the supernatant was discarded, and the pelletwas resuspended (2:1, vol/vol) with 10% SAS. An additional twelve hoursof agitation was followed by centrifugation at 3,000×g for thirtyminutes. The resulting supernatant was dialized using 10,000 molecularweight exclusion dialysis membranes against three changes of 0.025MTris/HCl, pH 7.5, (buffer A) for sixteen hours. Insoluble material wasremoved from the retentate by centrifugation (3,000×g for 30 minutes).

A series of agarose (triazine ring) immobilized textile dyes (Dye-MatrixScreening Kit, Amicon, Lexicton, Massachusetts) were prepared accordingto the manufacturer's instructions. Columns (9×32 mm, 2 ml bed volume)containing Matrix Gel Blue A (triozinyl dye Cibacron Blue 3GA), Red A(Procion Red HE3B), Blue B (Cibacron Brilliant Blue FBR-P), Orange A, orGreen A were equilibrated with 20 mM Tris/HCl (pH 7.5), then chargedwith 0.5 ml aliquots of the dialyzed retentate. Unbound material waseluted with 10 ml buffer A. The bound protein was eluted with 1.5M KClin buffer A. Eluent fractions were dialyzed overnight against buffer A,and protein concentration determined.

Eluents containing active material (Orange A-bound fractions) werefurther separated (10 ml/h, 4° C.) on a Sephadex G-50 (superfine) column(1.6×50 cm, V_(O) =60 ml, 5 ml/h, 4° C.) with buffer A. Elution profileswere determined using an ISCO absorbance meter at 254 nm. For estimationof molecular weight by gel filtration, the same Sephadex G-50 columnused for purification (2.6×70 cm, V_(t) =280, V_(O) =90 ml) wasequilibrated and developed with molecular weight standardsribonuclease-A, chymotrypsin, and ovalbumin in buffer A at 10 ml/h, 4°C. Fractions were then assessed for activity in the bioassay. K_(av) foreach standard and active fraction was calculated using V_(t) =280, V_(O)=90 (K_(av) =V_(e) -V_(O) /V_(t) -V_(O)).

Additional aliquots from the 10-55% SAS fraction of the hFF (10 ml) werepassed through a Concanavalin A-linked Sepharose 4B (Con A) column whichwas washed with 5 vol of 0.2M NaCl, 0.05M Tris/HCl (pH 7.4), thenfurther eluted with 0.2M α-methyl-D-mannoside in buffer A at a flow rateof 20 ml/h. Both Con A-bound and -unbound fractions were assessed foractivity in the bioassay. Chromatography fractions containing inhibitoryactivity were heated (56° C., 1 h) or trypsin digested (10 mg/100 ml)for three hours, and then retested for bioactivity.

Both Orange A-bound and 10-55% SAS hFF were further purified byisoelectric focusing using a Sephadex G-15 support matrix. The apparatusconsisted of a 4×30 cm water-jacketed glass column containing a 2.5×20cm G-15 Sephadex bed supported by a 2.5×8 cm Teflon elution plug under a25-m Millipore filter. The column was previously equilibrated withtwo-bed volumes of a solution containing carrier ampholytes (4% of pH3-10 and 4% of pH 2-4) in 12.5% glycerol. Cytochrome C was used as aninternal marker protein (pI=10.5). The fractions were then washed intothe column with 20 ml ampholyte-glycerol solution. A second Milliporefilter was placed on the top of the Sephadex bed. A 10-ml polyacrylamidesolution of 14% acrylamide, 0.3% Bis, and50μ1N,N,N',n'-tetramethylethylenediamine (polymerized by the addition of0.1 ml of 10% ammonium persulfate) was then poured over the filter. Uponcompletion of polymerization (twenty minutes), the column was inverted,the Teflon plug was removed, and a second acrylamide plug was layeredover the bottom filter. After polymerization of the bottom plug, thecolumn was returned to its upright position and lowered into anodebuffer containing 1% sulfuric acid. The remaining upper portion of thecolumn was filled with 1% ethanolamine. The column was cooled byrecirculating water at 1-4° C. throughout the procedure. Isoelectricfocusing was initiated at 800 constant V (16 mA) and allowed to proceedto equilibrium as monitored by an eventual decline in the milliamperageto 2.5 mA (8-12 h). Thereafter, pooled column fractions (3 ml) weredialyzed against buffer A to remove ampholytes before bioassay.

Since an apparent isoelectric point was reproducibly determined inmaterial isolated from the human ovarian vein blood by ampholytedisplacement chromatograph in Example One, this procedure was employedwith the 10-55% SAS-dialyzed hFF. Pooled aliquots (10 ml) were layeredonto a Polybuffer Exchanger 94 adjusted to pH 7.4 with 0.025M Imidazolebuffer, 10 ml/h, 4° C. The column was eluted with Polybuffer 74 adjustedto pH 4.0 with 1N HCl. Fractions that eluted at a pH greater than 7.4were collected and rechromatographed in the same Pharmalyte columnreequilibrated to pH 9.4 with ethanolamine 0.025M, and eluted withPolybuffer 96.sup.™ adjusted to pH 6.0 with 1N KOH.

The rats used in the bioassay were hypophysectomized and implanted asdescribed in Example One, and serum estradiol-17β concentration wasdetermined as described previously.

Control determinations (no injected test fractions) for unstimulatedovarian weight were 34.7±3.2 (SEM) mg/rat and for LH-FSH-stimulated were192.0±30.5 mg/rat. Control levels for trunk serum estradiol were12.5±0.7 pg/ml for unstimulated rats, and 118.5±21 pg/ml forLH-FSH-stimulated. Where indicated, 100% inhibition equals ovarianweight and/or serum estradiol concentration of mean unstimulated controlvalues. Zero percent inhibition equals ovarian weight or serum estradiolconcentration of LH-FSH-stimulated control rats. These results aresimilar to those which have been obtained with the previous applicationof this bioassay procedure in Example One. Tests of statisticalsignificance wer preformed by Student's test and Duncan's multiple rangeanalysis.

Protein separation was performed using a Waters HPLC/GPC Model 244Liquid Chromatograph equipped with a 0.75×50 cm TSK 3000 SW gelexclusion column. A 100 -μl aliquot of the dialyzed, Orange A-dye-matrixeluent was separated on each high performance liquid chromatographic(HPLC) run. The proteins were eluted from the TSK column using anisocratic gradient of 0.02M PBS (pH 7.0) at a flow of 0.5 ml/min. Theprotein peaks were detected by absorbance at 280 nm with a Watersvariable wavelength detector (Model M-450) and molecular weightestimates of the specific follicular fluid proteins were performed usinghighly purified molecular weight chromatography standards ofribonuclease-A, chymotrypsin, ovalbumin, and bovine serum albumin (BSA).

Table 1 summarizes the results of Dye-Ligand matrix chromatography ofthe 10-55% SAS-dialyzed hFF fraction. Although Orange A bound only 17%of the charged protein, 1.5M KCl eluted bioactive material thatcontained the greatest inhibition of the hMG-induced rat ovarian weightgain (89±6.8% SEM; P<0.05) when compared to the other bound fractions.

                  TABLE 1                                                         ______________________________________                                        FF Protein Recovery and Inhibition of Rat Ovarian                             Weight in Response to Exogenous Gonadotropin                                  Timulation by the 10-55% SAS Pooled hFF Fraction                              Developed through Dye-Matrix Chromatography                                   Unbound              Bound                                                            % Protein %          % Protein                                                                             %                                        Dye-Matrix                                                                            Recovered Inhibition.sup.a                                                                         Recovered                                                                             Inhibition.sup.a                         ______________________________________                                        Control 96        68 ± 3.2                                                                              4.6     0                                        Blue A  35        10 ± 4.7                                                                              63      0                                        Blue B  87        11 ± 8.1                                                                              21      14 ± 3.2                              Red A   38        0          63      0                                        Orange A                                                                              86        0          17      89 ± 6.8.sup.b                        Green A 31        0          68      0                                        ______________________________________                                         Bound material was eluted with 1.5 M KCl in TrisHCl (0.025 M. pH 7.5)         .sup.a Values are the means ± SEM.                                         .sup.b Significantly greater inhibition of rat ovarian weight response to     gonadotropin stimulation compared to other Dyematrix eluents (P > 0.05.       Student's t test).                                                       

FIG. 5 depicts the chromatographic elution profile of hFF developedthrough Sephadex G-50 at 254 nm after SAS 10-55% cut, dialysis (<10,000molecular weight), and elution of Orange A-bound material with 1.5M KCl.Fractions (2 ml) were tested in the LH-FSH-stimulated hypophysectomized,immature, DES-treated rat for inhibition of ovarian weight, and serumestradiol concentration (mean±SEM). An initial peak in absorbance can beseen at V_(e) -V_(O) ratio of 1.0-1.1, which after descending, reaches agradual ascending second plateau (V_(e) -V_(O) of 1.58-1.68). Biologicalactivity was determined as inhibition of ovarian weight and trunk serumestradiol levels. The same column was then equilibrated with molecularweight standards and developed with buffer A, allowing for molecularweight estimation (K_(av) =log V_(e) -V_(O) /V_(t) -VO) for fractionscontaining inhibitory activity. Molecular weight of eluents containinginhibitory activity was estimated to be 13,000-18,000.

Eluents from the isoelectric focusing of hFF after SAS (10-55%),dialysis and Orange A dye matrix chromatography (1.5M KCl eluate) (FIG.6) were evaluated for activity in the bioassay. Only fractions in the pHrange of 3.5-4.5 contained clear inhibition of ovarian weight and trunk17β-estradiol levels (not shown). When isoelectric point determinationwas performed using ampholyte displacement chromatography (FIG. 7),inhibition of rat ovarian weight was found in the pH range 3.5-4.5. Inaddition, a second area of inhibition in the bioassay was noted at (pH6.0-6.5.).

FIG. 8 depicts the HPCL elution profile of the Orange A-bound extractedhFF material eluted through a TSK 3000 analytical molecular weightexclusion column. FIG. 9 indicates the elution profile of the samematerial eluted through a TSK 3000 preparative molecular weightexclusion column. Each of these elution profiles displays uniquechromatographic patterns of FRP.

In FIG. 8, the HPLC eluent was divided into ten fractions based on peakabsorbances [0: 18.5-21.0 minutes (void volume); 1: 21.0-23.0 minutes;2: 23.0-25.0 minutes; 3: 25.0-28.5 minutes; 4: 28.5-32.2 minutes; 5:32.2-34.0 minutes; 6: 34.0-40.0 minutes; 7: 40.0-45.0 minutes; 8:45.0-49.0 minutes; 9: 49.0-53.0 minutes]. Aromatase inhibiting activityelutes at 40-45 minutes. These fractions correspond to the followingmolecular weight ranges: 1,100,000; 2, 100,000; 3, 70,000-100,000; 7:5,500-18,000; 8: 2,500-5,500; 9, 2,500. The retention times of peakabsorbances after HPLC elution, when correlated to molecular weightstandards, were highly reproducible (13 runs).

When HPLC fractions were tested in the bioassay (see FIG. 10),inhibition of ovarian weight gain and serum estradiol elevation wereevident in rats injected with the 5,500-18,000 molecular weight fraction[78±8% (SEM) and 89±10%, respectively (P<0.01)]as compared to the otherfractions. FIG. 11 shows a polyacrylamide gel, with sodium dodecylsulfate, of the fractions shown in FIG. 8 after HPLC. Columns 1-5 inFIG. 11 correspond, respectively, to the columns 3-7 in FIG. 8. The FRPis shown at the bottom of column 5, with a molecular weight of 15,000 to16,000 when compared to the molecular weight standards to the right ofcolumn 5. The standards include ribonuclease A at 14,000, andchymotripsinogen A at 22,000.

In Example One, there is described a protein, in the venous drainage ofthe human ovary which contains the preovulatory follicle, whichsuppresses the follicular response to gonadotropins. That this proteinwas not secreted in large amounts by anovulatory ovaries was evidencedby failure of the bioassay to detect inhibitory activity in the venousdrainage of the contralateral ovary in ovulatory patients as well asboth ovarian vein effluents from anovulatory women. In the presentexample, a comparable isolation procedure has shown a similar biolgicalactivity in human follicular fluid (hFF). The rat bioassay employed toidentify material in hFF which inhibits LH-FSH-mediated follicularstimulation was the same as that reported for human ovarian vein bloodextracts. Follicular inhibitory activity in hFF had a molecular weight,determined by HPLC size exclusion chromatography (10,000-18,000) that issimilar to that of follicular inhibitory activity recovered from humanovarian vein serum (14,000-17,000). The indicated isoelectric point forinhibitory activity in human ovarian vein extract, as determined bychromatofocusing (pH 5.8-6.4), is similar to the hFF extract reportedhere.

The follicular inhibitory substance reported here was derived fromfollicular aspirates of women during spontaneous ovarian cycles whosefollicles were hyperstimulated by clomiphene and hCG therapy.Consequently, no conclusion can be drawn regarding this material innormally developing follicles. However, since a similar activity hasbeen identified in the ovarian vein serum draining the spontaneouspreovulatory ovary (Example One) these data indicate this inhibitoryprotein to be a product of the dominant follicle itself.

EXAMPLE THREE Gonadal Regulatory Protein Fractions in Porcine FollicularFluid

Example Two details the isolation of a protein fraction (FRP) from humanfollicular fluid, which suppresses follicular response to gonadotropins.The present example employs an identical isolation, bioassay and HPLCprocedures with respect to porcine follicular fluid (pFF). Isoelectricfocusing demonstrated inhibitory activity at about pH 4.2-4.5 and pH6.27 and peak activity was found in the molecular weight range12,000-18,000 daltons.

Granulosa cells were isolated from rat ovaries and FSH binding wasdetermined by the procedure of Erickson, as described in Example Four.Aromatase activity of the rat granulosa cells was determined in aprocedure similar to that described therein.

Table 2 summarizes the results of dye-ligand matrix chromatography ofthe 10-55% saturated ammonium sulfate porcine follicular fluid fraction.Although Orange A bound only 13% of the charged protein, 1.5M KClelution recovered bioactive material which contained the greatestinhibition of hMG-induced rat ovarian weight response (p<0.05) whencompared to the other dye-ligand eluents. When these same fractions weretreated with heat (56° C.) or trypsin (10 mg %), no suppression inovarian weight was found.

                  TABLE 1                                                         ______________________________________                                        Protein Recovery and Inhibition of Ovarian                                    Weight Response from Dyematrix Chromatography                                 of the 10-55% Saturated Ammonium Sulfate                                      Dialyzed Porcine Follicular Fluid Fraction                                    Unbound             Bound                                                                       %                  %                                        Dye-Matrix                                                                            % Protein Inhibition                                                                              % Protein                                                                              Inhibition                               ______________________________________                                        CONTROL 94        78 ± 6.1                                                                             8.8      0                                        BLUE A  34        22 ± 8.3                                                                             65       0                                        BLUE B  81        0         20       39 ± 7.4                              RED A   33        12 ± 4.2                                                                             65       0                                        ORANGE  88        0         13       84 ± 7.4                              GREEN A 37        0         63       22 ± 3.8                              ______________________________________                                         *Significantly greater inhibition (p. 05 Student's T) of hMGinduced rat       ovarian weight response compared to other dyeligand eluents.             

FIG. 14 depicts the chromatographic elution profile of pFF at 280 nmdeveloped through Sephadex G-100 following the 10-55% SAS cut, dialysis,and elution of Orange A-bound material with 1.5M KCl. An initial peakcan be seen at a V_(e) /V_(o) of 1.1 to 1.17, thereafter trailing,followed by a gradual ascending second plateau at V_(e) /V_(o) 1.7 to1.9. When biological activity was assessed in the HIFR-hMG bioassay, 95%inhibition of both ovarian weight and trunk serum estradiol-17-B levelswere found at V_(e) /V_(o) of 1.5. The same column was then equilibratedwith molecular weight standard and developed with 0.025M PBS, pH 7.4,allowing for estimation of the K_(av) (V_(o) -V_(e) /V_(e) -V_(o)) offractions containing inhibitory activity.

When eluents from the isoelectric focusing (IEF) of the Orange A boundpFF extraction (FIG. 12) were evaluated for bioactivity by rat bioassay,only fractions within the range of pH 3.7 to 4.0 contained obviousinhibition of both rat ovarian weight response and trunk serumestradiol-17B levels (83.6±9.4% and 47.2% respectively). Serialdilutions of the extracted PFF fractions eluted from the Orange Adye-matrix with 1.5M KCl recovered from the isoelectric focusing columnwere tested for activity in the hypophysectomized immature DES-treatedfemale (HIFR-rat) bioassay. A dose response relationship was apparent(FIG. 13) with fractions in the pH 3.5-4.0 range, while other pH rangesfrom the IEF column were without inhibitory activity.

When aliquots of this Orange A matrix column were separated by hydrogenion chromatography (chromatofocusing), fractions corresponding to pH4.0-4.5 (FIG. 15, fraction 7) and pH 6.0-6.5 (FIG. 15, fraction 4)inhibited microsomal aromatase activity. When fractions 4 and 7 arefurther purified by HPLC molecular weight separation, aromataseinhibitory activity (FRP) is present in the fractions corresponding to15,000-16,000 daltons.

When aliquots from the ammonium sulfate 10-55% cut eluted from theOrange A dye matrix were passed through Concanavalin-linked Sepharose 4B(eluted 3×Vo with PBS followed by 0.2M alpha-methyl mannoside), noinhibitory activity in the rat bioassay was noted in the Con A boundfraction (i.e. eluted with mannoside) with only marginal recovery ofactivity (20-30% inhibition of ovarian weight response) in the materialeluted in the unbound PBS fraction (data not shown).

FIG. 16 depicts the HPLC elution profile of the Orange A-bound extractedPFF material after separation through the Sephadex G-100 column (V_(e)/V_(o) 1.3-1.7). The HPLC eluent was divided into five fractions basedon peak absorbances (1: retention time --25.0-27.8 minutes; 2: 27.8-31.0minutes; 3: 31.0-36.0 minutes; 4: 36.0-39.0 minutes; 5: 39.0-43.0minutes). These fractions corresponded to the following molecular weightranges: 1: 100,000-74,000; 2: 74,000-36,000; 3: 36,000-18,000; 4:18,000-12,000; 5: 12,000-5,800. The retention times of peak absorbancesafter HPLC elution, when correlated to molecular weight standards, werehighly reproducible (five runs).

When HPLC fractions were tested in the bioassay (see FIG. 17),inhibition of ovarian weight gain was evident in rats injected withfraction 3 (74±8%; p<0.01) and fraction 4 (51±9%, p<0.05) as compared tothe other three fractions. Inhibition of androstenedione conversion tototal immunoreactive estrogen by granulosa cells harvested from theHPLC-fraction treated rat's ovaries (n=6 ovaries/HPLC fraction) wasevident in HPLC fraction 3 (11.1±3 pg/10,000 cells/ml). When no testfractions were injected into the HIFR-hMG bioassay rat prior to thearomatase assay, a range of 30-60 pg of total immunoreactive estrogen/10,000 cells/ml was present in the control incubates.

FIG. 18 shows the results of FSH binding studies performed on thegranulosa cells removed from the HIFR-hMG rats used in the bioassay ofthe Sephadex-G100 fraction which contained inhibitory activity. Nodifference in FSH binding to the rat granulosa cells was evident betweenthe control and inhibitor treated HIFR-hMG rats.

Subsequent procedures, identical to those described in this exampleregarding porcine follicular fluid, have isolated FRP having a molecularweight range of about 12,000 to 18,000 daltons and an isoelectric pointof from pH 4.0 to 6.5, and inhibiting aromatase activity as describedabove, from bovine follicular fluid.

EXAMPLE FOUR Identification of Regulatory Protein(s) in Human GranulosaCell Secretions

Follicular fluid was aspirated from regularly menstruating women(twenty-five to thirty-five years old), undergoing clomiphene citrate(150 mg/day for five days, beginning three-eight days after the onset ofspontaneously occurring menses) and hCG (4000 IU, 36 hours beforeaspiration) therapy during participation in an in vitro fertilizationprotocol. Serum was collected daily, and estrogen concentrations weredetermined. When serum estrogen concentrations exceeded 800-1000 pg/ml,patients underwent laparoscopy for aspiration of follicles in excess of20 mm in diameter. Follicular aspirates were immediately centrifuged(800×g), granulosa cells were removed for culture, and the supernatantwas frozen. Follicular aspirates from seven patients were evaluated.

The aspirated follicular fluid volume, number of viable granulosa cells,and follicular fluid steriod concentrations from the largest folliclerecovered from the seven patients are depicted in Table 3. All of theantral fluids contained high concentrations of progesterone (7-12μg/ml), indicating premature luteinizations of the follicles (30-32) asa result of the clomiphene/hCG therapy. Approximately 100,000 viablegranulosa cells were obtained from each follicle

                  TABLE 3                                                         ______________________________________                                        Follicular fluid aspirate                                                     Pat- Vol-   Viable    Steroid conc (mg/ml)                                    ient ume    granulosa Estra-                                                                              Es-  Proges-                                                                              17-Hydroxy-                           No.  (ml)   Cells     diol  trone                                                                              terone progesterone                          ______________________________________                                        1    12.0   1.0 × 10.sup.5                                                                    396   37   12,377 212                                   2    7.1              204   3    11,761 889                                   3    12.2   2.6 × 10.sup.5                                                                    440   220  12,746 82                                    4    12.0   1.7 × 10.sup.5                                                                    296        10,132 176                                   5    5.3    0.75 × 10.sup.5                                                                   327        7,500  667                                   6    5.3    0.7 × 10.sup.5                                                                    1,740 411  9,197  1,091                                 7    8.8    10.5 × 10.sup.5                                                                   708   492  11,371 1,200                                 ______________________________________                                    

Individual hFF samples were slowly thawed and fractionated by dropwiseaddition of an equal volume of saturated ammonium sulfate duringpersistent agitation at 4° C. After a twelve hour incubation at 4° C.,the precipitate was recovered by centrifugation and resuspended (2:1,vol/vol) in 10% ammonium sulfate. An additional twelve hours of mixingwas followed by centrifugation at 3,000×g for thirty minutes. Theresulting supernatant was dialyzed (10,000 molecular weight exclusionmembranes) against phosphate-buffered saline (PBS) (0.025M; H 6.8) forsixteen hours at 4° C. and then lypholized. The retentate (500 mg in 0.5ml aliquots) was passed through a column (9×32 mm; bed volume, 2 ml)containing agarose-immobilized Orange A (Dye matrix, Amicon), which hadbeen equilibrated with 20 mM Tris-HCl, pH 7.5. Unbound material waseluted with 10 ml 20 mM Tris-HCl, pH 7.5 and bound material was elutedwith 10 ml 1.5M KCl in 20 mM Tris-HCl pH 7.5. Bound eluent fractionswere dialyzed overnight against PBS or distilled water. Proteinconcentrations were determined by the method of Lowry et al., J. Biol.Chem. 143:265.

Rats were prepared for bioassay as described in the previous samples.

Results of control determinations (no injected test fractions) were34.8±3.2 mg/rat for unstimulated ovarian weight and 122.0±13.5 mg/ratfor FSH-stimulated ovarian weight. Control levels of trunk serumestradiol were 12.5±0.7 pg/ml for unstimulated and 118.5±21 pg/ml forLH/FSH stimulated. Where indicated, 100% inhibition equals the ovarianweight and/or serum estradiol concentration of mean unstimulated controlvalues. Zero percent inhibition equals the ovarian weight or serumestradiol concentration of LH/FSH-stimulated control rats. These resultsare similar to those obtained during the previous examples. Tests ofstatistical significance were performed by Student's t test and Duncan'smultiple range analysis.

FIG. 19 depicts the effect of extracted human follicular fluid (ehFF)and respective granulosa cell culture media (24, 48, and 72 hours) onthe inhibition of LH/FSH (2 IU)-stimulated rat ovarian weightaugmentation and serum estradiol secretion (2 ml/rat). Each valuerepresents the mean±SEM of three rats.

All four follicular fluid extracts (ehFF) contained inhibitory activity,as evidenced by inhibition of both rat ovarian weight (40-65%) and trunkserum estradiol concentrations (85-100%). Bioassay of culture media fromthe respective granulosa cell cultures collected twenty-four,forty-eight and seventy-two hours after plating indicated thatinhibitory activity was present during the first forty-eight hours ofincubation. Variability in the initial twenty-four hour determination ofpatients 2 and 3 may relate to initial plating efficiency. Importantly,all bioassay determinations were made after complete medium changes eachtwenty-four hours. No inhibitory activity was noted after seventy-twohours of culture.

FIG. 20 depicts the compiled (mean±SE) bioassay results of culture media(containing FRP) derived from granulosa cell cultures of patients 5, 6and 7. All media tested without gonadotropins added to the culturecontained inhibitory activity of both ovarian weight and trunk serumestradiol concentrations exceeding 75% throughout the first two days ofculture. Thereafter (days 3 and 4), the inhibitory activity of mediafrom unstimulated cultures declined (43% and 37%, and 18% and 12% forovarian weight and serum estradiol levels, respectively). Aftercoincubation of the granulosa cells with varying doses of LH/FSH (0.3,1.0 and 3.0 U), inhibitory activity, as determined in the HIFR-hMGbioassay, was markedly suppressed in all cultures after the firsttwenty-four hours (>20%).

When progesterone concentrations were determined for these culturemedia, an inverse correlation between inhibitory activity in thebioassay and culture medium progesterone concentrations was apparent.The unstimulated cultures (no added LH/FSH) had the least progesteronethroughout the four days of culture (<10 ng/ml), but containedinhibitory activity (FIG, 20), albeit in decreasing amounts as cultureduration continued. However, after LH/FSH was added to the granulosacell cultures in increasing amounts, the progesterone concentrationsrose in a dose-and time-dependent pattern, while bioassay inhibitoryactivity declined to essentially the limits of bioassay detectability(FIG. 20).

High performance liquid chromatography

The high performance liquid chromatographic (HPLC) separation offollicular fluid steroids was performed.

Protein separation was performed using the same Waters HPLC/GPC Model244 liquid chromatograph equipped with two Waters I-125 gel exclusioncolumns connected in series. A 10-μl aliquot of the dialyzed, Orange Adye matrix-bound eluent was separated on each HPLC run. The proteinswere eluted from the I-125 columns using an isocratic gradient of 0.02MPBS, pH 7.0, at a flow of 0.5 ml/minute (800 psi). The protein peakswere detected at 280 nm, and molecular weight estimates of the specificfollicular fluid proteins were performed using highly purified molecularweight chromatography standards of ribonuclease-A, chymotrypsin,ovalbumin, and BSA.

Granulosa cells were cultured for up to four days in twenty-four hourintervals using 35×10-mm tissue culture dishes and 2 ml medium 199containing 25 mM Hepes supplemented with 100 U/ml penicillin, 100 μgstreptomycin sulfate, and 15% fetal calf serum (medium A). Cultures weremaintained in a humidified, 95% air-5% CO₂ incubator at 37° C. Aftereach twenty-four hour incubation period, spent medium was collected andstored frozen at -20° C. until bioassay was performed. Where indicated,human menopausal gonadotropin (FSH-LH, 1:1) was added to specificcultures at the time of complete medium change. At the end of eachtwenty-four hour incubation period, spent medium was collected forbioassay, and 2 ml fresh medium were added. The final cell pellets weredispersed in medium A, and aliquots (0.5 ml) of the cell suspension werediluted with 0.05 ml trypan blue for quantitation of viable cells in ahemocytometer. Initial plating density was 0.5×10⁵ granulosacells/plate.

Granulosa cells were isolated from rat ovaries and were collected bycentrifugation at 800×g at 4° C. for ten minutes. FSH binding wasdetermined using a modification of known techniques. Rat FSH, providedby the National Pituitary Agency, was labeled by the chloramine-Tprocedure. Cells were resuspended in appropriate volumes of PBS-01%gelatin (PBS-gel), pH 8.0. All assays were run with three concentrationof labeled hormone (100 μl), buffer (PBS-0.1% gel; 100 μl) and 100 μlcells. Reactions were initiated by the addition of granulosa cells andwere carried out for four hours at 25° C. Reactions were terminated byadding 1 ml cold PBS, followed by centrifugation at 30,000×g for tenminutes. The supernatant was carefully aspirated, and the pellet wasrewashed with 1 ml PBS. The final pellet was counted in a γ-counter.Specific binding was calculated as the difference between binding in thepresence (nonspecific) and absence (total) of an excess of unlabeledhormone.

FIG. 21 depicts the FSH binding studies performed on the granulosa cellsremoved from the HIFR-hMG-treated rat ovaries used in the bioassayexperiments shown in FIG. 19. Specific binding of rat FSH (rFSH) togranulosa cells was determined by incubating three concentrations oflabeled rFSH in the presence and absence of excess unlabeled rFSH. Ratgranulosa cell specific FSH binding was similar whether the rat receivedinjections of spent medium from human granulosa cell cultures or saline.However, a marked difference in the ovarian weight and trunk serumestradiol concentrations of these rats was present.

Replicate 0.1-ml portions of each rat granulosa cell suspension werepipetted into 12×75-mm polystyrene tubes. Androstenedione, the referentaromatase substrate, was added in 0.1 ml medium A (final concentration1.0×10⁻⁷ M). All incubations were performed in triplicate for threehours at 37° C. in a shaking water bath (120 cycles/minute). Thereaction was stopped by transferring the tubes to an iced water bathbefore centrifugation for five minutes at 1000×g. The supernatants weredecanted and stored at -20° C. until measurements of estradiol andestrone were performed. Control incubations (no androstenedione added)were processed in the same way. Blank estrogen values obtained for thecontrols were subtracted from the corresponding values for incubationsin the presence of androstenedione. Aromatase activity was expressed asestrogen production (nanograms per viable granulosa cell).

Rat granulosa cell aromatase activity is shown in FIG. 22 and wasmarkedly inhibited (P<0.01) by treatment with spent media from all fourdays of culture (2.4±0.4, 2.9±0.7, 2.4±0.4, and 2.1±0.2 pgestrogen/10,000 viable cells/ml, respectively, compared to salinecontrol 4.6±0.2 pg estrogen/10,000 viable cells/ml. Controldeterminations were performed on rat granulosa cells collected fromHIFR-hMG-treated rats which did not receive culture medium injections.Inhibition of relative estrogen production by rat granulosa cells in thepresence of 10⁻⁷ M androstenedione was seen throughout all four days ofhuman granulosa cell culture medium treatment. Taken together, thesedata indicate that although no marked inhibition of rat granulosa cellFSH binding was induced by the human granulosa cell culture medium, aclear disruption of aromatase activity occurred, which may account forthe decreased rat ovarian weight and serum estradiol concentrations.

Using the same purification techniques and bioassays described above, asimilar protein or proteins have been identified in the culture mediaderived from the granulosa cells of human follicles. This data indicatesthat human granulosa cells secrete a protein that inhibits follicularresponse to gonadotropins.

All of the antral fluid's steroid concentrations suggested prematureluteinization. After extraction, all seven follicular fluids containedinhibitory activity, as evidenced by reduction of both rat ovarianweight (45-85%) and trunk serum estradiol concentrations (85-100%) inthe HIFR-hMG bioassay. Bioassay of these follicles' granulosa cellculture media indicated inhibitory activity present during the firstforty-eight hours, while no inhibitory activity was noted afterseventy-two hours of culture. Spent culture media derived from granulosacells cultured without additional gonadotropins contained inhibitoryactivity in the HIFR-hMG bioassay throughout the first two days invitro. Thereafter (days 3 and 4), inhibitory activity of media fromunstimulated cultures declined. After coincubation of the granulosacells with varying doses of LH/FSH, inhibitory activity was markedlysuppressed even after the first twenty-four hours. An inversecorrelation was apparent between inhibitory activity in the bioassay andthe culture medium progesterone level. Although FSH binding of granulosacells derived from rats used in the HIFR-hMG bioassay was similar withor without injection of test fractions, their aromatase activity wasmarkedly inhibited by treatment with human granulosa cell culturemedium. These data indicate that although no marked inhibition in ratgranulosa cell FSH binding was induced by the human granulosa cellculture medium, a clear disruption of aromatase activity occurred, whichaccounts for the decreased rat ovarian weight and serum estradiolconcentrations found in the bioassay. Disruption ofgonadotropin-mediated aromatase induction by an intrafollicular proteinmay, in part, modulate the local balance between C-19 steroid aromataseand 5-α-reductase enzymic activities in individual follicles.

A variety of nonsteroidal regulators of ovarian function have beenidentified in a variety of species, including oocyte maturationinhibitor, luteinizing inhibitor, folliculostatin or inhibin, and FSHbinding inhibitor. The biophysical characteristics of the intragonadalprotein of this invention are different from such substances. Thismaterial elutes through gel exclusion chromatography with a molecularweight of 12,000-18,000, binds to Orange A dye matrix, and has anapparent isoelectric point of from pH 4.0-4.5 to 6.0-6.5. Theseobservations indicate that in addition to the intrafollicular steroidalmileau a variety of nonsteroidal compounds, secretory products of thegranulosa cells or other ovarian compartments, contribute to theregulation of folliculogenesis.

EXAMPLE FIVE The presence of the gonadal regulating protein in Testis

Bovine testis were homogenized and the homogenate precipitated with 55%saturated ammonium sulfate. The precipitate was resuspended and dialyzedagainst distilled water. The retentate was eluted though Sephadex G-100with TRIS buffer (20 mM, pH 7.5). The eluant corresponding to amolecular weight range of 12,000-18,000 was collected and charged intoan Orange A dye matrix column. The column was eluted with TRIS buffercontaining 0.5M KCl (20 mM, pH 7.5). This eluant contained FRP asevidenced by the inhibition of porcine granulosa cell aromatase activity(FIG. 23; bTEc and bTE-OAB columns) and rat serum estradiol levels (FIG.24; BTE+FSH and BTE-OAB+FSH columns). Further, this activity wascontained in testicular extract fractions with isoelectric points offrom pH 4.1-4.5 and 6.0-6.5. This demonstrates that the testis and itssecretory product, rete testis fluid, also contains FRP.

EXAMPLE SIX

Extraction of FRP from Sertoli Cell Cultures

Granulosa cells secrete FRP, and Sertoli cells are the embryonichomologue in testis of the granulosa cells in the ovary. Further, thebiological activities of granulosa cells are present in the Sertolicells insofar as they have been identified and are relevant to gonadalfunction. Thus, the Sertoli cell provides an additional, readilyavailable source of FRP. Accordingly, Sertoli cells may be grown in cellculture and the recovered culture media is extracted by the proceduresdescribed in the foregoing examples, principally salt participation,chromatographic procedures and electrophoresis.

II. Biological Activity EXAMPLE SEVEN The Effect of the GonadalRegulatory Protein as an Aromatase Inhibitor

In Examples One through Six, protein(s) in human ovarian venouseffluent, human and porcine follicular fluid, bovine testis fluid andspent media from human granulosa and rat Sertoli cell cultures inhibitedrat ovarian weight gain in response to gonadotropin stimulation. Gonadalfluid extracts containing this protein were found to have a molecularweight of 12,000-18,000 and an isoelectric point of from about pH4.0-4.5 to pH 6.0-6.5. As inhibin, another protein secreted by humangranulosa cells, increases with follicular maturation and decreases withluteinization, individual human follicles from untreated as well as hMGand clomiphene treated women were assessed for FRP activity, and thatactivity was correlated with the follicles' follicular fluid steroid andinhibin concentrations.

Follicular aspirates were obtained from women (agedtwenty-four-thirty-two years) who were participating in an in vitrofertilization protocol. All patients had regular ovulatory menstrualcycles based on monthly vaginal bleeding and at least a single lutealphase serum progesterone in excess of 3 ng/ml. When serialultrasonographic examinations (ADR Model 2140 real-time sector scannerwith a 3.5 mHz rotating head transducer) demonstrated a folliculardiameter in excess of 18 mm laparoscopy was performed for aspiration ofall follicles greater than 16 mm in diameter. Follicular aspirates wereimmediately transferred to an adjacent laboratory for removal ofgranulosa cells by centrifugation (600×G, 15 minutes) and storage offollicular fluid (-37° C.) until assay. Follicular fluid concentrationsof estradiol, progesterone, 17-hydroxy progesterone, androstenedione andtestosterone were determined by established radioimmunoassay techniques.

FRP was isolated from individual follicular fluid samples substantiallyas described in Example Two.

Aromatase Activity

Porcine granulosa cells were collected from fresh ovaries obtained atthe local slaughterhouse. After washing in serum free HAMS-HEPES tissueculture media, 5×10⁵ cells in 0.2 ml of medium were pipetted into 12×75mm polystyrene tubes. Triplicate 200 μl portions of each follicularfluid preparation at three different protein concentrations (700-10μg/ml) were tested. Each tube then received 100 μl of FSH (10 g) inculture medium and was incubated at 37° C. in a shaking water bath forthree hours. An atmosphere of 90% N₂, 5% O₂ and 5% CO₂ was maintainedthroughout the incubation. The incubation was stopped by the addition of0.5 ml Hams-Hepes media and centrifugation at 1000×g for five minutes.The granulosa cells were then resuspended in 0.5 ml Hams-Hepes media,whereupon 100 μl of cells were assayed for armoatase activity.Androstenedione, the referent aromatase substrate, was added in 0.1 mlmedium (final concentration, 1.0×10⁻⁷ M). All incubations were performedin duplicate for three hours at 37° C. in a shaking water bath (120cycles/minute). The reaction was stopped by transferring the tubes to aniced water bath before centrifugation (five minutes at 1000×g). Thesupernatants were decanted and stored at -20° C. until measurements ofestrogen were performed. Control incubations (no androstenedione added)were processed in the same way. Blank estrogen values obtained for thecontrols were subtracted from the corresponding values for incubationsin the presence of androstenedione. Aromatase activity was expressed asestrogen production (nanograms per viable granulosa cell).

Inhibin Activity

Ten percent (weight/volume) activated charcoal (Norite A) was added tothe individual follicular fluids, and stirred continuously overnight at4° C., followed by centrifugation (1000×G, twenty minutes) and sterilefiltered to remove the charcoal. The charcoal-stripped follicular fluidcontained 10 pg/ml of androstenedione, progesterone, and estradiol asdetermined by radioimmunoassay. Inhibin activity was determined usingthe degree of inhibition of basal (i.e. non-LHRH stimulated) twenty-fourhour FSH secretion by dispersed rat anterior pituitary cells in primarymonolayer. For each cell culture, anterior pituitary glands wereobtained from 20 cycling female Sprague-Dawly rats (250-300 gm bodyweight). During the thirty minute interval required for removal of allthe pituitary glands, each gland was placed in medium (pH 7.4, 20° C.).Pituitary glands were finely minced with scissors and incubated in amixture containing 1% viokase, 3.5% collagenase, and 3% bovine serumalbumin in medium buffer at 37° C. for thirty to forty-five minutes. Thedispersed cells were counted using a hemocytometer and pituicyteviability was determined by 1% trypan blue exclusion. Typically, morethan 90% of the dispersed cells were viable. The cells were diluted to aconcentration of 2.5×10⁵ viable cells per ml growth medium. Growthmedium consisted of HAMS F10 containing 10% fetal calf serum withpenicillin, fungiezone and streptomycin (50 μ/ml and 50 mg/ml and 50mg/ml respectively). Cells were added to tissue culture dishes in avolume of 2.0 ml growth media, and attachment of the cells to the wellsurface was completed by two days. After cell attachment, the originalgrowth media was discarded and the cells were washed twice withadditional HAMS balanced salt solution. Thereafter, three concentrationsof charcoal-treated follicular fluid (0.02%, 0.2%, 2%) were added to theplate. A lyophilized porcine follicular fluid standard (PFF1 KT-1,provided by CP Channing) was resuspended in water and tested in eachassay at 0.003%, 0.016%, 0.08%, 0.04%, and 2% concentrations.Twenty-four hours later, the spent culture media was assayed induplicate using the NIH-RIA kit for rFSH.

Data Analysis

The mean estrogen concentration in the serum control tube for each FRPassay was set at 100%, and the response in each test was expressed as apercentage of the control estrogen concentration. The coefficient ofvariation for each group of three replicate tubes was calculated. If thecoefficient was 15%, the estrogen assay was repeated and/or one value ofthe three was discarded. A curve was constructed in which the percentinhibition of estrogen in each well was plotted vs the proteinconcentration of follicular fluid added. Unknown values were determinedby plotting the experimentally determined values at three differentprotein concentrations (700-10 μg/ml) on a log-linear graph andextrapolating the value at 50 μg/ml. The percent inhibition of estradiolat 50 μg of unknown follicular fluid was read off the standard curve andexpressed as percent aromatase inhibition for that follicle.

For the inhibin assay, the response in each test plate was expressed asa percentage of the control FSH concentration, which was set at 100%.The coefficient of variation of each group of three replicate plates wascalculated. If the coefficient was 15% the assay was repeated. Astandard curve was constructed in which the percent inhibition of FSH instandard wells was plotted vs the log of the standard added. Leastsquares linear regression was used to construct a standard curve in thelinear portion of the dose response curve. Unknown follicular fluidinhibin values were determined by plotting the experimentally determinedvalues (0.02%, 0.2%, 2%) on a log-linear graph and extrapolating thevalue at 1%. The percent inhibition of rFSH at 1% of unknown follicularfluid was read off the standard curve and expressed as Channing units (1CU=1 unit of inhibin standard=the inhibition of rFSH in rat pituicyteculture by 1 nl of charcoal treated, ethanol extracted porcinefollicular fluid).

Tests for statistical significance were performed by one-way analysis ofvariance and Duncan's new multiple range test. Correlation betweenfollicular fluid steroid concentration and FRP activity was performedusing regression analysis with tests of statistical significanceperformed by t test corrected for N.

Patient Outcome

Seven patients underwent follicular aspiration during an untreatedspontaneously occurring ovarian cycle. At the time of laparoscopy, onlyone antral follicle was seen in each patient which was aspirated. Ninepatients received clomiphene citrate therapy (150 mg/day, cycle days5-9), providing a total of twenty-four follicles with diameters >16 mm.All except one patient had multiple follicles aspirated. Six patientswho underwent hMG therapy (150 IU LH/150 IU FSH administered dailybeginning on cycle day 3 until follicle aspiration), providedtwenty-three follicles. Care was taken to aspirate all follicles withdiameters in excess of 16 mm. There was no difference between treatmentgroups in follicle size which averaged 18.7±0.9 mm (x±SEM, range 16-24mm). At the time of aspiration, serum estradiol levels averaged 1456pg/ml±285 pg/ml for all patients studied (range of 310-3200 pg/ml) withno significant difference between treatment groups.

Validation

To establish the number of porcine granulosa cells for the FRP assay,the following porcine granulosa cell concentrations were used: 0.5×10⁶,1×10⁶, 2×10⁶ cells/ml. The total amount of estrogen produced following athree-hour incubation was 55±9, 140±27, 375±48 pg/culture dish,respectively. Consequently, 2×10⁶ porcine granulosa cells were used ineach subsequent assay. To evaluate the effects of porcine FSH (NIH P-2reagent) the follicular protein (100 μg), porcine granulosa cellcultures were prepared with or without porcine FSH added to the media(0.5 ml final volume), incubated at 37° C. for three hours in a shakerbath with 95% O₂ and 5% CO₂, after which androstenedione (10⁻⁶ M) wasadded in 0.5 ml of growth media. Cultures were incubated for three hoursat 37° C. in a shaker bath then centrifuged (1000×G for fifteen minutes)and media collected for estrogen determination. Without FSH, theestrogen production was 439±41 pg/ml; with FRP added, the estrogenproduction was 22.4±41.7 pg/ml. When 2 units/ml of porcine FSH wereadded without FRP 728 pg/ml estrogen were produced. Addition of FRPproduced a dose-response relationship 1000 μg FRP: 200.26 pg/ml; 200 μgFRP: 306.37 pg/ml; 50 ng FRP: 345.41 pg/ml; 10 μg FRP: 334±18 pg/ml ofestrogen. Accordingly, individual patient values were extrapolated to 50ng/ml of FRP for comparisons of activity.

The FRP in elution profiles from Orange A were analyzed using threedifferent concentrations of KCl: 0.17M KCl yielded 3.1 mg protein/mlwhich had a 37% inhibition of aromatase, 0.5M KCl eluted 6 mg protein/mlwhich had an 84% inhibition of aromatase and 1.5M KCl eluted 0.05 mgprotein/ml which had a 6.7% inhibition of aromatase. Accordingly, the0.5M KCl fraction was used to elute the active material from the OrangeA bound column. KCl (0.5M) was found to have no effect in the granulosacell aromatase assay: control (without KCl, 3 determinations, 710±41ng/ml. with KCl, 712±38 ng/ml). Duration of incubation time was assessedwith and without FSH. Two hour assay incubations yielded 2 ngestrogen/ml; twenty-four hours: 5.3 ng estrogen/ml. With 2 ng FSH added,39 ng estrogen/ml at two hours and 6 ng FSH/ml produced 8 ng estrogen/mlat twenty-four hours. With 1 ng FSH/ml, 35±1.8 ng estrogen/ml wereproduced at two hours and 4.7±0.9 ng estrogen/ml at twenty-four hours.Accordingly, a three-hour incubation was used to determine specific FRPactivity.

When FRP preparation was heated to 56° C.×1 hours, the inhibition ofgranulosa cell aromatase was lost. The FRP activity levels (% inhibitionof porcine granulosa cell aromatase by 50 μg of follicular fluid) foruntreated patients was 14.16±5.32% (X±SEM). For patients receiving hMG,FRP

Correlation of FRP vs Follicular Fluid Steroids

There was a positive correlation between follicular fluid estradiolconcentrations and FRP protein activity in untreated patients (r=0.689,p.<0.01). For patients receiving hMG therapy, there was no significantcorrelation between FRP activity and follicular level estradiolconcentrations (r=0.490, <0.1). For patients receiving clomiphenetherapy, the correlation between FRP activity and follicular fluidestradiol was described by two populations using a second degreeregression analysis (r₂ =0.853, p<0.01). Correlation between follicularfluid progesterone concentrations and FRP activity for untreatedpatients (r=0.622, p<0.05) and hMG treated patients (r=0.756, p<0.005)was significant. For clomiphene treated patients, correlation betweenfollicular fluid progresterone and FRP activity were not significant.The correlation between follicular fluid 17-hydroxyprogesterone valuesand FRP activity for the untreated (r=0.833, p<0.001), as well as hMG(r₂ =0.853, P<0.0025) and clomiphene (r₂ =0.637, p<0.025) treatedpatients was significant by second order regression analysis. Thecorrelation between FRP and androstenedione concentration from untreated(R=0.241), hMG (r=0.357), and clomiphene (R=0.219) treated patients wasnot significant, nor was the correlation between testosterone and FRPactivity significant (r=0.477, 0.409, 0.480, respectively). The averagelevels were 18.09±3.46%, and for patients receiving clomiphene,13.7±5.36%. There was no statistically significant difference in thevalues between the three treatment groups. The amount of estradiol inthe follicular fluid of untreated patients was 2.59±1.2 μg/ml, forpatients receiving hMG therapy, 0.34±0.5 μg/ml, for patients receivingclomiphene, 1.31±0.34 μg/ml. These values were all significantlydifferent (p<0.05, untreated vs clomiphene, clomiphene vs hMG; p<0.01untreated vs hMG). Progesterone values for untreated patients were9.84±3.35 μg/ml, for hMG-treated patients, 5.18±1.1 μg/ml, and forclomiphene-treated patients, 11.3±2.3 μg/ml. These difference weresignificant for the unstimulated and hMG-treated patients (p<0.05) andhMG vs clomiphene treated patients (p<0.01). The 17-hydroxyprogesteroneconcentrations for patients receiving no additional therapy were1.66±0.25 μg/ml, for those receiving clomiphene therapy 2.6±0.3 μg/ml,and for those receiving hMG therapy: 0.76±0.11 μg/ml. All these valueswere significantly different (hMG vs clomiphene p<0.01; hMG vsunstimulated p<0.01; unstimulated vs clomiphene p<0.25). Follicularfluid androstenedione concentrations in untreated patients were 61.9±43ng/ml. For hMG and clomiphene treated patients they were 85.5±37, and84.8±43 ng/ml, respectively. Follicular fluid testosterone levels fromuntreated patients were 7.34 ng/ml±3.7. For the treated patients, therewas no difference in the follicular fluid testosterone concentrations inpatients receiving either hMG (7.09±2.14 ng/ml) or clomiphene (6.14±1.8ng/ml). inhibin activity for untreated patients was 50±1.9 CU. Inpatients receiving treatment, inhibit activity was 8.2±2.3 and 35.4±3.7CU for clomiphene and hMG treatment, respectively. Differences betweenhMG and either clomiphene or untreated patients were highly significant(p<0.001). Correlation between inhibin and FRP activities for untreatedpatients was significant (r=0.654; p<0.05; FIG. 26). However, there wasno statistically significant correlation between inhibin and FRPactivities in patients receiving either hMG (r=0.270) or clomiphene(r=0.262).

These observations report the presence of an aromatase inhibitor (FRP)in a purified fraction of human follicular fluid. This follicular fluidprotein fraction (FRP) has previously been shown to inhibit hMG-mediatedincreases in rat ovarian weight and serum estradiol concentrations. Asis shown hereinafter in Example Thirteen, when this protein fraction isinjected into regularly menstruating monkeys, it disruptsfolliculogenesis resulting in either anovulatory cycles or luteal phaseinsufficiencies accompanied by low serum estradiol and progesteroneconcentrations without markedly altered peripheral serum gonadotropinlevels. This data, taken together with those presented previously,suggest that the developing granulosa cell, through production of anaromatase inhibitor, is capable of autoregulating the estrogenproduction of its own and other follicles.

EXAMPLE EIGHT Gonadal Regulatory Protein Inhibition of MicrosomalAromatase Activity

In this example, the effect of the gonadal-regulating protein onaromatase activity was studied in cell-free placental microsomepreparations which were prepared in accordance with the techniquesdescribed in Mol. Cell. Endocrinol., 6 (1976) pp. 105-115 and J. Clin.Endocrinol. Metab., 39 (1974) 754-760.

Aromatase incubations were carried out in a total volume of 0.6 ml in12×75 ml glass tubes as described in the above-identified references.Incubations contained 0.3 ml of placental cell-free preparation inbuffer A, 0.10 ml of ¹⁴ C-androstenedione (A, 10⁻⁶ molar), NADPH (10⁻⁶molar) and nicotinamide (0.4 molar) in buffer A. Gonadal regulatoryprotein test fractions (0.2 ml, 12 to 18 kd, pI 4.0-6.5), werepreincubated with placental extracts (20 minutes) and then the ¹⁴C-androstenedione-NADPH mixture was added. Reaction intermination/quenching was performed by addition of 100-fold excessunlabeled A. Estrogen concentrations were determined by radioimmunoassayas described in J. Clin. Endocrinol. Metab., 39, 754-760.

FRP-dose response determinations were performed using a three-minutereaction time. The velocities determined over a range of substrateconcentrations (0.5-2 mM) demonstrated Michaelis-Menton type kineticswith a Km of 0.8 mM as shown in FIG. 27. The aromatase velocities weredetermined over a range of FRP concentrations (0, 62.5, 125, 250, 500and 1,000 μg/ml). The use of Dixon kinetic plotting techniquesdemonstrated that FRP inhibited microsomal aromatase with an AppKi=3×10⁻⁵ M (FIG. 28).

These date demonstrate the non-competitive inhibition of FRP onaromatase activity. Enzyme inhibition may be of three types. First,competitive inhibition describes the competition of the inhibitor forthe substrate-specific site on the enzyme. Second, non-competitiveinhibition describes a direct inhibition of the enzyme withoutcompetition for the substrate-specific sites. Third, uncompetitiveinhibition describes an indirect inhibition of the enzyme by additionalmechanisms. In FIG. 28, the intersection of the lines at the abscissa,as opposed to ordinal intersection (competitive) or non-intersection(uncompetitive) show that FRP is a non-competitive inhibitor ofaromatase activity. FRP directly interacts with the aromatase moleculeto inhibit its activity.

EXAMPLE NINE Modulation of Beta-ol-Dehydrogenase (3β-ol) Activity ByFollicular Protein

Porcine and human granulosa cells from medium sized follicles (2-5 mm indiameter) were cultured (100,000 cells per culture) with variousconcentrations of FRP (12-18 kd. pI 4.0-6.5) isolated from follicularfluid. To these cultures pregnenolone (10⁻⁵ M) and either hCG of pFSHwere added. The conversion of pregnenolone to progesterone was used todetermine 3-beta-ol-dehydrogenase activity in the granulosa cells. FRPcaused a biphasic response in progesterone production. Gonadalregulatory protein in the concentration of 167 μg/ml caused a 10 foldincrease in progesterone production, while the 500 μg/ml concentrationcaused a return to baseline levels. These results are shown in FIGS. 29and 30. Although pFSH induced a dose response increase in progesteroneproduction, hCG produced no change in progesterone levels. Low doses ofthe gonadal regulatory protein acted synergistically with low doses ofpFSH to increase 3-beta-ol-dehydrogenase activity. However, high dosesof FRP inhibited the low dose pFSH stimulation of3-beta-ol-dehydrogenase activity. High doses of pFSH (10 μg/culture)overcame both the low dose enhancement and the high dose inhibition ofFRP on 3-beta-ol-dehydrogenase activity. Kinetic analysis of FRPmodulation of 3-beta-ol-dehydrogenase activity was performedsubstantially as described in Example Eight. The effects of FRP on3-ol-dehydrogenase activity in human placental microsomes are of anon-competitive nature.

EXAMPLE TEN Inhibition of LH/hCG Receptors In Granulosa Cells by FRP

FRP (12-18 kd., pI about 4.0-6.5) was isolated from porcine follicularfluid substantially as described in Example Three.

Granulosa Cell Cultures

The granulosa cells were counted using a hemocytometer and viability wasdetermined by 1% trypan blue exclusion. Cells were cultured (2×10⁵) in 2ml of Medium 100 containing 10% fetal calf serum with penicillin andstreptomycin (100 g/ml and 100 U/ml respectively) in 12×75 mm Falconplastic test tubes. FSH (10 ng), and/or FRP (1 mg) were added at theinitiation of culture. Media was changed after seventy-two hours.

Binding Analyses

Porcine granulosa cells were suspended in appropriate volumes ofPBS-0.1% gelatin (PBS-gel). All assays were run with five concentrationslabeled hCG (10 μl), buffer (PBS-0.1% gel, 100 μl), and cells. Reactionswere initiated by the addition of granulosa cells and were carried outfor four hours at 25° C. All reaction tubes were precoated with 5% BSAto reduce non-specific absorption. Reactions were terminated by addingone ml of cold PBS followed by centrifugation at 30,000×g for tenminutes. The supernatant was carefully aspirated and the pellet rewashedwith one ml of PBS. The final pellet was counted in a gamma counter.Specific binding was calculated as the difference between binding in thepresence (non-specific) and absence (total) of an excess of unlabeledhormone. Data were analyzed by Scatchard plots. Duplicate determinationswere performed in three separate assays at each time interval.

Follicular protein significantly reduced porcine granulosa cell hCGbinding by seventy-four hours of culture. This effect was prevented withthe co-administration of FSH.

By ninety-six hours of culture, no change in porcine granulosa cell hCGbinding was apparent with FSH, follicular protein, or both compared tocontrol cultures demonstrating cellular recovery after removal offollicular protein at seventy-two hours of culture.

Thus, in addition to regulating key enzymatic steps in the steroidogenicpathway (aromatase, 3β-ol dehydrogenase), general granulosa cellresponse to trophic LH stimulation is also mediated by follicularprotein through inhibition of LH receptor function.

EXAMPLE ELEVEN Adenylate Cyclase Activity

The effects of FRP on FSH-induced adenylate cyclase activity in porcinegranulosa cells was evaluated using Gpp (NH)p and forskolin aspharmacological probes of adenylate cyclase activity. With the additionof 100 μg/ml of FRP (12-18,000 pI 4.0-6.5), a significant decrease inthis activity was found. Maximal inhibition of cAMP formation wasachieved with 1 mg/ml of FRP. Adenylate cyclase activity reached amaximum 20 min after incubation with FSH and returned to baseline by 45minutes. FRP induced a parallel reduction in adenylate cyclase activityduring this same interval of time (FIG. 31). Adenylate cyclase activityin the membranes of FRP+FSH and FRP alone treated cells wassignificantly less than in cells incubated with FSH (p<0.05). Adenylatecyclase activity of FRP treated cells was unchanged in the presence ofmethyl-isobutyl-xanthine. Further, when FRP was heated (56° C., 45 min.)or precipitated with 10% TCA, it lost the capability to inhibitadenylate cyclase. The 50% inhibitory dose (ID50) for FRP inhibition ofGpp(NH)p stimulated adenylate cyclase activity with preincubation ofgranulosa cells with FSH was 350 μg/ml and 80 μg/ml without FSH. TheID50 for the FRP inhibition of forskolin stimulated adenylate cyclaseactivity was 350 μg/ml (FIG. 32). Adenylate cyclase activity wasdetermined after a 10 min. incubation with forskolin or Gpp(NH)p. Whenthese responses were compared during a 5-20 min. interval, the Gpp(NH)pstimulated adenylate cyclase activity was more sensitive to inhibitionby FRP than forskolin stimulated adenylate cyclase activity (p<0.05).Adenylate cyclase activity stimulated by Gpp(NH)p was also, on a molarbasis, more sensitive to FRP inhibition than forskolin stimulatedactivity, FRP had no apparent effect on LH or hCG responsive adenylatecyclase activity in these granulosa cell preparations. No othernaturally occurring substance has previously been shown to selectivelyinhibit FSH responsive adenylate cyclase activity in granulosa cellswithout also inhibiting LH responsive adenylate cyclase activity. Inconclusion, these data demonstrate that FRP inhibited FSH responsiveadenylate cyclase activity in porcine granulosa cells.

EXAMPLE TWELVE Reduction of Follicular Atresia

Sheep have been injected intramuscularly with FRP (2 mg at 08:00 and16:00 hours) for fourteen days. During this interval, there was adestruction of the estrous cycle such that they did not ovulate. On Day14 of treatment the ovaries were removed, fixed in formalin, seriallysectioned (4 microns) and the sections were mounted on glass slides formicroscopic evaluation after staining with hematoxylin and eosin. It wasevident that FRP had inhibited ovulation by blocking the normaldevelopment process whereby a developing follicle either ovulates ordegenerates by becoming atretic. This was evident in development of thefollicles. These findings demonstrate that the normal life span of thetotal follicular pool could be significantly prolonged by thetherapeutic administration of FRP. With prolongation of the life span ofthe follicular pool, a prolongation of the reproductive capacity of anindividual would naturally follow. Since the alteration in function andcomposition of sex steroid-dependent structures, which is commonlyreferred to as menopause in females, would be prevented or forestalledtotally, or in part, if the sex steroid secretion was maintained, andsince the ovarian follicles are the source of such sex steroids, itfollows that prolongation of the life span of ovarian follicles by FRPtherapy will lead to prevention or reduction of the clinicalmanifestations of the menopause.

III. Whole Animal Studies EXAMPLE THIRTEEN Inhibition of the PrimateOvarian Cycle by FRP

The Examples heretofore presented report the identification of a heatand trypsin labile protein extracted from porcine, bovine and humangonadal fluid which inhibited ovarian response to gonadotropins. Theactivity of this protein, secreted by human granulosa cells, increasedwith increasing follicular fluid estradiol levels and decreased withincreasing follicular fluid progesterone levels (as shown in ExampleFour) both in vivo and during granulosa cells' luteinization in vitro(Example Four). This material has also been found to inhibit granulosacell aromatase activity in both porcine (Example Three) and human(Example Four) granulosa cells in vitro. Follicular fluid extractscontaining this activity are shown in said Examples to have a molecularweight of 12,000-18,000 and an isoelectric point of about pH 4.0-4.5 to6.0-6.5, showing that the biophysical nature of this protein is notinhibin. In the present Example, the effects of this follicular proteinfraction on the integrated hypothalamic-pituitary-ovarian axis of thenormally cycling monkey are assessed.

Adult female rhesus monkeys (Macaca mulatta; n=8) were selected becauseof reproductive characteristics indicating normal ovarian function andmenstrual regularity. The FRP employed in this Example was theinhibitory follicular fluid protein fraction which was obtained asdescribed in Example Three.

Five monkeys were treated with FRP extracted from porcine follicularfluid and three monkeys served as vehicle controls. The FRP (3 mg in 1ml of 0.01M PBS, pH 7) was administered (IM) at 07:00 hours and 19:00hours beginning on day 1 of the menstrual cycle for a total oftwenty-nine injections. Total dose for each monkey was 87 mg. Controlmonkeys received only PBS over the same interval. Iron supplement wasadministered once each week. Daily (09:00-11:00 hours), femoral bloodsamples (3.5 ml) were collected beginning with the onset of menses(day 1) and were continued until the onset of next menses.Radioimmunoassays for LH, FSH, 17ρ-estradiol, and progesterone in serumwere performed.

Monkeys which received FRP injections had either no serum LH peak norelevation of serum progesterone (n=2, FIG. 33), or a midcycle LH surgefollowed by an inadequate luteal phase as demonstrated by low serumprogesterone levels and/or early onset (twenty-four days intermenstrualinterval) of vaginal bleeding (n=2, FIG. 34). One FRP treated monkey hada midcycle LH surge followed by depressed follicular phase estradiol andluteal phase serum progesterone levels (not shown). The 95% confidencelimits for the vehicle control and other values obtained for thispopulation are depicted by the shaded area in FIGS. 33 and 34. Serumestradiol levels were reduced throughout the interval of FRP treatmentin the monkeys without LH surges in the late follicular phase (FIG. 34)and in those with luteal phase defects (FIG. 33). Serum FSH and LHlevels were within the 95% confidence intervals for FRP treated monkeys(FIG. 33). However, in all five FRP treated monkeys, serum FSH levelsrose during the course of therapy. In the monkeys with inadequate lutealphases (FIG. 33), serum estradiol levels were below the 95% confidenceintervals in the late follicular and mid luteal phases. While the serumlevels of both estradiol and progesterone were markedly suppressed afterthe LH surge. In subsequent cycles (N=3/monkey) onset of vaginalbleeding occurred in the usual twenty-six--thirty day monthly intervaland no toxic effect of GRP treatments was noted.

FRP administered to normally cycling monkeys throughout the follicularphase of the menstrual cycle reduce peripheral estradiol levels withoutmarkedly affecting FSH, resulting in either apparent anovulation orinadequate luteal phases. The mixed response in serum sex steroid levelsin the FRP treated monkeys may reflect a variable ovarian sensitivity toFRP. That serum FSH levels were not significantly inhibited indicatesthat this material has a biological activity different from that ofinhibin activity in charcoal-extracted whole porcine follicular fluid.Further, when FRP was tested in an inhibin assay, its activity waseither at the limits of sensitivity or undetectable. In contrast to theinhibition of FSH, normal or rising serum FSH levels during FRPtreatment were found. This indicates that the purification of follicularfluid described herein removed the major inhibin activity. The molecularweight of the described FRP is less than 20,000 while inhibin activityhas been associated with molecular weights greater than 45,000.

The in vivo observations of reduced serum estradiol levels in GRPtreated monkeys support the previously described Examples of granulosacell aromatase inhibition by both human and porcine derived FRP.Further, these observations are in agreement with examples of reducedserum estradiol levels in FRP treated rats.

Luteal phase defects, as evidenced by suppressed serum estradiol andprogesterone levels and early onset of vaginal bleeding, result frominadequate follicular maturation in the preceding follicular phase.Taken together with the observations reported in this Example of reducedserum estradiol and progesterone levels associated with FRP treatment,these data indicate that the FRP described in the present invention hasa direct intraovarian action which disrupts the normal process offolliculogenesis by an action apart from gonadotropin stimulation.

EXAMPLE FOURTEEN Inhibition of Spermatogenesis

Male mongrel dogs were injected with 2 mg of FRP derived from porcinefollicular fluid (12-18 kd., pI 4.0-6.5), as described in Example Three,at 08:00 and 16:00 hours for 20 days. On Day 20 of therapy the testiswere obtained, fixed in formalin, sectioned by microtome (four micronsections), mounted on slides and stained with hematoxylin and eosin.Upon evaluation of the slides, a marked reduction in mature spermatozoawas present in the seminiferous tubules in the FRP-treated dogs ascompared to the controls. Moreover, there was an 87% reduction inpacytene spermatocytes and a 44% reduction in mature spermatogonia inthe FRP-treated dogs.

IV. Preparation of FRP Antibodies EXAMPLE FIFTEEN

Preparation of FRP Antibodies

Antibodies (monoclonal and polyclonal) to FRP and its cogeners andanalogs may be prepared for diagnostic and therapeutic uses includingbut not limited to fertility control. Antibody in this contex refers toa synthetic protein which binds FRP and alters FRP biological activity.

Antibodies to FRP are prepared by either polyclonal or monoclonaltechniques:

Polyclonal: 5 adult rabbits are immunized with 0.1 mg of FRP suspendedin complete Freund's adjuvant (5 ml). One ml of this preparation isinjected subcutaneously at 20 different sites in the back and neck. Thisis followed by monthly injections thereafter. Ear vein phlebotomies areperformed after each monthly booster injection and the sera obtained arechecked for titer, affinity, and specificity.

In a specific example, 1 μg of FRP (from Example Three) was solubilizedin 0.5 ml physiological saline and emulsified with an equal volume ofFreund's adjuvant to prepare an inoculum.

New Zealand White, female rabbits weighing 2-1/2-3 kg were bled via themedian ear artery for pre-immune serum. A 10×20 cm area on the back wasshaved, then each rabbit was intradermally injected at multiple points.Approximately 50-75 μl of inoculum was injected into 10-25 sites in theshaved area; rabbits 1 and 3 received 0.6 ml and 1.6 ml, respectively.The rabbits were boosted in similar fashion six weeks later receiving0.5 ml and 1.5 ml, respectively. Six and ten day post-boost, the rabbitswere test bled, again through the median artery. Sera containing thepolyclonal antibodies thus obtained were titred via RIA against ¹²⁵I-labelled FRP as follows:

Rabbit sera were two-fold serially diluted in RIA buffer from 1:1000 to1:64,000. 100 μl each of the RIA buffer, diluted serum, and ¹²⁵ I-GRPand 200 μl RIA buffer were also prepared. The tubes were incubated atroom temperature overnight. One-half ml (0.5 ml) precipitating solutioncontaining 2% goat-antirabbit gamma globulin and 4% polyethylene glycolin RIA buffer was then added to each tube except the total tubes. Thetubes were vortexed, incubated at room temperature for ten minutes, thencentrifuged at 3000 rpm for an additional ten minutes. The supernatantwas aspirated and the precipitin-pellet counted for one minute on agamma counter. The rabbit sera was found to contain antibody which bound¹²⁵ I-FRP (12 to 18 kd., pI 4.0-6.5) in all dilutions tested, including1:64,000.

Monoclonal: BALB/c mice are immunized with FRP by intraperitonealinjection (25 μg)×2. Thereafter, the spleens are collected and cellsuspensions prepared by perfusion with DMEM. The BALB/c spleen cells arefused with SP 2/0-Ag 14 mouse myeloma cells by PEG and the resultanthybridomas grown in HAT selective tissue culture media°30% fetal calfserum. The surviving cells are allowed to grow to confluence. The spentculture media is checked for antibody titer, specificity, and affinity.

Specifically, the mice were immunized with FRP (from Example Three)adjuvant emulsion described above. Each mouse first received 0.2 ml ofthis emulsion intraperitoneally, then were reinjected in similar fashionwith 0.1 ml six weeks later. Mouse serum was obtained ten days after thesecond injection and then tested for anti-FRP activity via ELISA. Themouse exhibiting the highest absolute anti-FRP activity was chosen forcell fusion.

Three to four days prior to fusion, the chosen mouse was intravenouslyinjected with 0.1 ml FRP solubilized in physiological saline, andSP2/0-Ag14 BALB/c myeloma cells maintained in log phase culture. On theday of fusion, the mouse was sacrificed and its spleen asepticallyremoved. Spleen cell suspension containing B-lymphocytes and macrophageswere prepared by perfusion of the spleen. The cell suspension was washedand collected by centrifugation. Myeloma cells were also washed in thismanner. Live cells were counted and the cells were placed in a 37° C.water bath and 1 ml of 50% poly-ethylene glycol in DMEM slowly added.The cells were incubated in the PEG for one to one-and-a-half minutes at37° C., after which the PEG was diluted by the slow addition of media.The cells were pelletted and 35 to 40 ml of DMEM containing 10% fetalbovine serum was added. The cells were then dispensed into tissueculture plates and incubated overnight in a 37° C., 5% CO₂, humidifiedincubator.

The next day, DMEM-FCS containing hypoxanthine, thymidine, andaminopterin (HAT medium) was added to each well. The concentration ofHAT in the medium to be added was twice the final concentrationsrequired; i.e. H_(final) =1×10⁻⁴ M, A_(final) =4.0×10⁻⁷ M, and T_(final)=1.6×10⁻⁵ M.

Subsequently, the plates were incubated with 1×HAT medium everythree-four days for two weeks. Fused cells were thereafter grown inDMEM-FCS containing hypoxanthine and thymidine. As cell growth became1/2 to 3/4 confluent on the bottom of the wells, supernatant tissueculture fluid was taken and tested for FRP-specific antibody by ELISA.Positive wells were cloned by limiting dilution over macrophage orthymocyte feeder plates, and cultured in DMEM-FCS. Cloned wells weretested and recloned three times before a statistically significantmonoclonal antibody was obtained. Spent culture media from the chosenclone contained antibody which bound ¹²⁵ I-FRP (12-18 kd., pI 4.0-6.5)in all dilutions tested, including 1:64,000.

Each of the above described antibody containing solutions was testedagainst FRP secreted by granulosa cells and found to bind toindependently produced FRP. Antibodies from both polyclonal andmonoclonal preparations are screened for affinity by evaluating theability to inhibit the reduction of aromatase activity by FRP in humanplacental microsomes. Those antibodies which block this reaction weretitered by incubating various dilutions of the antibody with a fixedmass of radioactively labelled FRP in 100 μl of assay buffer (TRIS0.025M, pH 7.4) at 4° C. overnight with constant agitation. The dilutionof antibody that bound 50% of the labelled FRP under these conditionswas defined as the titer. Specificity was determined in the same manneras the above only column fractions not containing FRP activity wereradiolabelled and screened for antibody binding.

It will be apparent from the above description of FRP antibodies that awide variety of diagnostic tests is possible using the antibodies of theinvention. Techniques for the preparation and use of antibodies anddiagnostic test methods are known in the art. Since naturally-occurringFRP plays a central role in the ovulatory process, quantification of thelevel of FRP in body fluids provides diagnostic information ofsignificance. The overproduction of FRP by the gonads, an indication ofthe presence of a condition such as ovarian cancer, could easily bedetected in body fluids such as serum through the use of an immunoassaywhich employs the novel antibodies according to methods known in theart. Similarly, in attempting to diagnose causes of infertility, animmunoassay to detect decreased levels of FRP in the body would be auseful adjunct to known hormone assays. Further uses for the antibodiesinclude the induction of superovulation in livestock. The directadministration of FRP antibodies to sheep has been shown to inducemultiple ovulations in ewes.

More specifically, the isolation of FRP from porcine follicular fluid(12-18 kd., pI 4.0-6.5) has allowed the production of antiseracontaining antibody to FRP which cross-reacts with human FRP. Thisantibody may be used in an enzyme-linked immunosorbent assay (ELISA) andother assays suitable for quantitation of FRP in body fluids. In aparticular example, an ELISA assay was used to quantitate relative FRPimmunoreactivity in human serum in postmenopausal, normally menstruatingand anovulatory patients.

Polyclonal antibodies were prepared from porcine FRP (12-18 kd. fractionhaving isoelectric points in the range of pH 4.0-4.5 and 6.0-6.5)according to the method described above, and titered via RIA againstlabeled porcine FRP (having similar characteristics) in dilutions up to1:64,000.

A parallel-line dilution assay comparing the binding of the antibody toFRP in human serum to different antigen-containing samples by ELISA wasperformed with the following samples: porcine granulosa cell culturemedia, human granulosa cell culture media, human urine, bovine serumalbumin, ribonuclease A and chymotrypsinogen. Immunorecognition ofsamples in serially-diluted porcine and human granulosa cell culturemedia and urine showed parallelism, whereas those of bovine serumalbumin, ribonuclease A and chymotrypsinogen showed no binding, thusindicating a lack of recognition of the antibody for proteins in thosematerials.

The levels of FRP in the following patients was determined by ELISAassay. Ten patients undergoing ovarian extirpative surgery were all ofreproductive age, and had regular menses with no clinical or laboratoryevidence of ovarian dysfunction, and all underwent a total abdominalhysterectomy and bilateral salpingo-ophorectomy. Peripheral venous bloodwas obtained before surgery and twenty-four hours post-operative. Tenpost-menopausal patients were 53-63 years of age and requiredhospitalization for conditions unrelated to ovarian function. Thirteenovulatory patients, all under age 40, had problems unrelated to ovarianfunction including infertility due to tubal, cervical or male factor orprevious tubal sterilization. These patients had regular menstrualcycles which were documented as ovulatory based on serum progesteronelevels greater than 2 mg/ml. The anovulatory patients each had a historyof oligomenorrhea with chronic anovulation. The anovulatory patientsreceived 50-250 mg of chlomiphene citrate on menstrual cycle days 5-9for ovulation induction.

Using the ELISA-determined levels of FRP in the ovulatory women as abasis, post-menopausal women had significantly lower serum levels ofFRP, and similar levels were found in the serum of reproductive-agewomen twenty-four to forty-eight hours after ophorectomy, versussignificantly higher pre-operative FRP levels in this group.

Serum FRP levels at the 3rd to 5th menstrual cycle day differedsignificantly in two groups of anovulatory women. A first group had highlevels of serum FRP, and a second group very low levels approximatingthe levels of post-menopausal women. Both groups of anovulatory patientshad similar low peripheral estradiol levels. The first group having lowserum FRP levels comprised those with whom chlomiphene citrate therapywas successful in inducing ovulation. In contrast, the anovulatorypatients with significantly elevated serum FRP levels failed to ovulateafter chlomiphene therapy. Significant differences between these twogroups were also apparent twenty-two to twenty-three days after thebeginning of the last menstrual period. Elevated FRP levels in serumhave thus been shown to be useful in predicting which anovulatorypatients will or will not respond to chlomiphene citrate therapy.

As detailed above, FRP antibodies provide a wide variety of usefuldiagnostic procedures. A determination of the levels of FRP in mammalianbody fluids, or patterns of these levels over time, and the comparisonof these levels or patterns with predetermined normal data yieldssignificant diagnostic information. For example, in one aspect of theinvention FRP has been found to be substantially elevated in instancesof gonadal cancer, i.e., in the body fluids of patients having ovarianor testicular cancer. It should be noted that there presently exists noreliable diagnostic test for this condition. In addition, it has alsobeen found that varying levels of immuno-reactive FRP are found in urineduring active phases of follicle growth and during lutenization,allowing the determination of the activity and timing of ovarianfunctions with the FRP antibodies of the invention. Further, FRP levelsremain elevated during the conception cycles of pregnancy, as opposed toa pattern of luteal phase decline of FRP in non-pregnant women. Thus, acomparison of these patterns or levels provides a significant early testfor pregnancy.

As noted above, it has been demonstrated that FRP levels in human bodyfluids vary significantly during gonadal cycles and gamete development,and antibodies to FRP have been used to monitor and predict gonadalfunction. These data provide methods for the diagnosis of the cause ofovarian dysfunction and for the determination of the treatment foranovulatory patients.

In Example Sixteen, a specific monoclonal antibody was produced and usedin an ELISA assay to quantify and monitor human FRP levels in urine, andto establish a pattern of normal FRP levels for comparison with patternsobtained from individuals with putative ovarian dysfunction. Significantdiagnostic and therapeutic advantages are obtainable with this method,as will be apparent to one having skill in the art.

EXAMPLE SIXTEEN

A second monoclonal FRP antibody was prepared form FRP which waspurified essentially as described in Example Three above, and which ismore specifically described in "Biochemical and PhysiologicalCharacterization of Follicular Regulatory Protein: A Paracrine Regulatorof Folliculogenesis", Ono et al., Am. J. Obstet Gynocol. 1986,154:709-716, incorporated by reference herein. This protein exhibitedthe definitive biological effects of the inhibition of aromataseactivity, LH receptor function and ovarian response to gonadotropins andhad a molecular weight of about 15,000 (14-16 kd) and an isoelectricpoint in the range of from pH 4.5 to 6.5. Female BALB/c mice wereinjected with this purified FRP in a 1:1 suspension with completeFreund's Adjuvant according to standard techniques. Approximately 10⁸spleen cells and 2×10⁷ log phase myeloma cells (SP 2/0-Ag14) were mixedand pelleted at 900×g for five minutes. The cells were washed withmedium, re-pelleted and resuspended in 50% peg, recentrifuged anddispensed into microculture plates containing a macrophage feeder layerand fed every three days with HAT medium. The feeder cells were BALB/cmacrophages obtained by peritoneal lavage. Peritoneal exudative cellswere seeded and allowed to attach overnight before fusion.

After 10-14 days in culture, the supernatant fluids from each activehybridoma were screened by an ELISA. Of the 150 hybridomas originallypositive, 125 were expanded into 10 ml cultures and frozen. Of these,twenty-six continued to produce desirable antibodies. Hybridomasproducing antibody were subcloned by limiting dilution on feeder layersof spleen cells until all subclones were positive for production ofantibody against FRP. The colonies grown in wells seeded at one cell perwell were considered monoclonal and were tested for anti-FRP antibody bythe ELISA. Isotype determinations were obtained with the ELISA usingmouse isotype-specific rabbit antibodies and goat anti-rabbit antibodiescoupled to peroxidase all subclones were found to be of the IgC class byisotyping.

To assess the specificity of the antibody, aliquots of FRP were analyzedon polyacrylamide gels. Samples were then electrotransferred tonitrocellulose paper, fixed, blocked in buffered saline and BSA and thenincubated overnight with a 1:1,000 dilution of anti-FRP antibodies.Combined immunogold-silver staining and Auro Dye total protein stainingwas performed to visualize the bands. FRP immunoreactivity was measuredby a competitive ELISA. And the immunohistochemical localization of FRPwas accomplished by employing avidin biotinylated-peroxidase complex.

Control procedures were carried out on adjacent polyacrylamide gelsections, and the selected FRP antibody (identified as 138G3) wasreplaced by an irrelevant monoclonal antibody (IgMTepC183,Sigma). Thisantibody was diluted to give the same total protein concentration as the138G3 FRP antibody. The specificity of the antibodies for FRP wasverified by Western blot against FRP, follicular fluid and serum-freespent culture media from granulosa cells previously shown to contain FRPby bioassay. When purified fractions of FRP were injected into femalerats, the ELISA detected significant immunorecognition in serum samplesobtained one hour later compared to the controls. The bound andflow-through fractions from the immunoaffinity column were assessed forimmunoreactivity by the ELISA and for bioactivity by inhibition ofgranulosal conversion of androstenedione to estrogen. The immobilizedantibody (138G3) recognized FRP since the bound fraction from theimmunoaffinity column had a higher specific immunoreactivity (37.6±8IRU/ml) as well as bioactivity (27.5±4% inhibition of aromatase/ml)compared to the unbound fractions (undetected).

Quantitation of FRP in a variety of heterogeneous proteins by the ELISAassay using the 138G3 antibody was demonstrated. Serum from female andmale rats, human seminal plasma, bovine and human serum albumin, serumand urines from patients that were menopausal or underwent oophorectomyand spent media from porcine theca cell cultures were not recognized bythe assay. However, serial dilutions of equine, porcine, and humanfollicular fluid; spent media from human and porcine granulosa cellcultures as well as from metastatic human granulosa cell tumor, producedparallel dilution profiles.

The biotin-avidin complexed 138G3 monoclonal antibody appeared brown onhematoxylin-stained sections of ovaries. No brown staining was apparentwith the use of the conjugated reagents alone nor with the biotin-avidincoupled to the irrelevant IgM at a concentration of 50 μg/ml of protein.Specific staining with 138G3 was limited to the mural granulosa cellpopulation in follicles without histological evidence of atresia (i.e.,viable follicles). No thecal, stromal or cumulus cell staining wasapparent in viable follicles nor were any follicle cells stained inpreantral or primordial follicles. In contrast, follicles with evidenceof granulosa cell pyknosis (atretic follicles) contained enhancedstaining of all follicular epithelial cells.

The 138G3 antibody was employed in an ELISA assay to determine levels ofFRP in the urine of twelve regularly menstruating women over time. Theselevels, and the simultaneously-obtained serum levels of LH, FSH,estradiol and progesterone as well as ultrasonographic determination ofthe largest follicle diameter are shown in FIG. 35. Urinary levels ofFRP were detectable above baseline five days before the LH surge,continued to rise thereafter reaching maximum levels four to five daysafter the LH surge, and then declined through the late luteal phase.This pattern correlates with the decrease in the number of healthyfollicles during the pariovulatory and mid-luteal phases of themenstrual cycle. The lowest FRP levels occurred during the earlyfollicular phase, a time which is characterized by the largest number ofviable follicles during the menstrual cycle. Viable follicles are notlikely to be a predominant source of FRP in the luteal phase since theirnumber decreases during this time. During the luteal phase the number ofatretic follicles increases, especially those 4-6 mm in diameter. Thistemporal correlation between rising levels of FRP and an increase in thenumber of atretic follicles suggests that the corpus luteum may play arole in the induction of atresia.

Inappropriate exposure to elevated FRP levels could limit the ability offollicles to respond to FSH resulting in a suppressed follicularmaturation and estradiol production. A group of anovulatory patientswith serum gonadrotropin levels within the normal range was found tohave elevated levels of immunoreactive FRP in their peripheral serum. Adifferential ovarian response to clomiphene citrate therapy by theseanovulatory patients (as described above) was observed on days 11-12 ofthe menstrual cycle. There appeared to be follicular abnormalitiesduring the intercycle interval as demonstrated by reduced serumestradiol levels and concommitant elevations in serum FRP levels 3-5days after the onset of the last menstrual period.

Comparison of the above data with the control levels set forth in FIG.35, provides a determination of gonadal function, specifically thedetermination and treatment of anovulatory patients. For example,anovulation may result from an overproduction of FRP and subsequentreduction in follicular responsivity to gonadotropin stimulation. Thatadditional gonadotropin exposure was not successful in overcoming thisputative FRP-induced block in follicular maturation is evidenced by theelevated levels of FRP in other anovulatory patients 11-12 and 22-23days after the onset of the last menstrual period even after receivingclomiphene citrate therapy on days 3-7 of the cycle. Anovulatorypatients who responded to clomiphene citrate therapy by increasing theirperipheral estradiol levels on days 11-12 and presumably ovulated asevidenced by elevated serum progesterone levels on days 22-23 did nothave elevated FRP levels during these intervals. Thus, folliculardysfunction expressed clinically as anovulatory cycles can be properlyidentified as resulting from abnormalities in ovarian protein, or insteroid hormone secretion.

The invention in general in certain aspects in particular are broad inscope, for example, the proteinaceous substance of the invention may beproduced by other methods.

In particular, a c-DNA probe can be prepared against the biologicallyactive portion of FRP and used to identify the FRP genome in granulosacells or Sertoli cells from any mammalian species. The identified genomecan then by synthesized into a plasmid which can then be employed toproduce recombinant DNA in proliferating bacteria according to methodsknown in the art.

In addition, granulosa or Sertoli cells may be transformed, e.g. by SV40virus, to produce FRP in quantity.

In summary, the intragonadal protein, its identifying characteristics,methods of production and uses set forth herein identify a novel andunique proteinaceous substance. FRP is described herein to be the onlyproteinaceous substance known which inhibits aromatase activity bydetermination of the extent of the conversion of androgens to estrogens.Moreover, such inhibition is unique in that it is non-competitive inplacental microsomes. Further, FRP produces a biphasic effect on 3β-oldehydrogenase activity in cultured granulosa cells as shown in FIGS. 29and 30.

In addition, FRP inhibits FSH induction of LH receptor formation asdetermined by addition to an in vitro culture of granulosa cells, andinhibits FSH responsive adenylate cyclase activity as similarlydetermined.

In addition to this biological activity, FRP is identified by theelution profile through a molecular weight exclusion column under theconditions set forth in Example Two, as indicated by the designation"FRP" of the elution curve in FIG. 9. The elution profile through ahydrogen ion exclusion column, particularly the area designated by theindicated peaks numbers 4 and 7 in FIG. 15, further identifiesidentifying characteristics of FRP when conducted under the conditionsset forth in the Example Three. Moreover, a polyacrylamide gelelectrophoretic pattern from a high pressure liquid chromatographiccolumn, as shown by the designation "FRP" in column 6 of FIG. 11,uniquely identifies FRP when the pattern is obtained as set forth inExample Two.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of the invention and, withoutdeparting from the spirit and scope thereof, can adapt the invention tovarious usages and conditions. The regulatory protein described hereinis defined primarily by its biological effect in a biological system.This phrase is meant to define a reversible effect, that is, one thatdoes not involve the destruction of endocrine functions such as by theheating or other denaturation of proteins. For example, theadministration of the protein to a mammal inhibits aromatase activity,and the cessation of this administration allows the reversal of theinhibitory effect.

While the biological activities set forth herein define the protein, thephysical characteristics set forth also provide distinguishing features.The protein moiety which provides the described biological activityshows a wide range of molecular weights (from 5,500 to 18,000 daltons)and an electrophoretic range of from about pH 3.5 to about 7.0. Morepreferably, the physical characteristics of the protein are identifiedas ranging from a molecular weight of about 10,000 up to 18,000 daltons,and having an isoelectric point of from about pH 4.0 to about 6.5. Basedon the data set forth herein, the physical characteristics of theprotein moiety which produces the above-described antibody has amolecular weight of about 15,000 daltons and an isoelectric point ofabout pH 4.5 to 4.75. However, changes in form and the substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient; and although specific terms have been employed herein, theyare intended in a descriptive sense and not for purpose of limitation,the scope of the invention being delineated in the following claims.

What is claimed is:
 1. A monoclonal antibody which binds a gonadalregulatory protein, said gonadal regulatory protein being characterizedin that it:(a) inhibits intragonadal activity of aromatase, asdetermined by extent of conversion of androgens to estrogens; (b) doesnot affect normal intragonadal FSH level while inhibiting aromataseactivity; and (c) has a molecular weight of from about 5,500 to about18,000 daltons and an isoelectric point of from about pH 4.0 to about6.5 as determined by electrophoretic mobility or hydrogen ionchromatography.
 2. The antibody of claim 1 which is an IgG antibody. 3.The antibody of claim 1 which is an IgG antibody.
 4. The antibody ofclaim 1 characterized in that the protein to which it binds has amolecular weight of from about 10,000 to about 18,000 daltons.
 5. Theantibody of claim 1 characterized in that the protein to which it bindshas a molecular weight of about 15,000 daltons and an isoelectric pointof from about pH 4.5 to about pH 4.75.
 6. A polyclonal antiserum to agonadal regulatory protein, said gonadal regulatory protein beingcharacterized in that it:(a) inhibits intragonadal activity ofaromatase, as determined by extent of conversion of androgens andestrogens; (b) does not affect normal intragonadal FSH level whileinhibiting aromatase activity; and (c) has a molecular weight of fromabout 5,000 to about 18,000 daltons and an isoelectric point of fromabout pH 4.0 to about 6.5 as determined by electrophoretic mobility orhydrogen ion chromatography.
 7. The antiserum of claim 6 characterizedin that said at least one antibody is an IgG antibody.
 8. The antiserumof claim 6 characterized in that said at least one antibody is a murineIgG antibody.
 9. The antiserum of claim 6 characterized in that theprotein to which said at least one antibody binds has a molecular weightof from about 10,000 to about 18,000 daltons.
 10. The antiserum of claim6 characterized in that the protein to which said at least one antibodybinds has a molecular weight of about 15,000 daltons and an isoelectricpoint of from about pH 4.5 to about pH 4.75.
 11. A monospecific fractionof a polyclonal antiserum to a gonadal regulatory protein, saidmonospecific fraction comprising at least one antibody which binds tosaid gonadal regulatory protein, said gonadal regulatory protein beingcharacterized in that it:(a) inhibits intragonadal activity ofaromatase, as determined by the extent of conversion of androgens toestrogens; (b) does not affect normal intragonadal FSH level whileinhibiting aromatase activity; and (c) has a molecular weight of fromabout 5,500 to about 18,000 daltons and an isoelectric point of fromabout pH 4.0 to about 6.5 as determined by electrophoretic mobility ofhydrogen ion chromatography.
 12. The monospecific fraction of claim 11characterized in that said at least one antibody is an IgG antibody. 13.The monospecific fraction of claim 11 characterized in that said atleast one antibody is a murine IgG antibody.
 14. The monospecificfraction of claim 11 characterized in that the protein to which said atleast one antibody binds has a molecular weight of from about 10,000 toabout 18,000 daltons.
 15. The monospecific fraction of claim 11characterized in that the protein to which said at least one antibodybinds has a molecular weight of about 15,000 daltons and an isoelectricpoint of from about pH 4.5 to about pH 4.75.